Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-Jun-2013 to 01-Nov-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and 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
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-Jun-2013 to 01-Nov-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and 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.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adapted 27th July 1995
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Recognized by international guidelines as a recommended test system.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Animals: Rat, RccHanTM: WIST(SPF)
Breeder: Harlan Laboratories, B.V., Netherlands
Number of Animals: 48 males: 12 per group
48 females: 12 per group
Age (at Start of Treatment): Approximately 11 weeks
Body Weight Range (at Start of Treatment): Males 329 to 382 g, females 185 to 236 g
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. 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.

HUSBANDRY:
Conditions:
Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 18 ± 3 °C from 13-Jun-2013 to 26-Jun-2013 and 22 ± 3 °C from 27-Jun-20113 onwards; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, which was considered not to have any influence on the study. The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle

Accommodation:
Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment. 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 Harlan Teklad 2018C rodent maintenance diet was available ad libitum.

Water:
Community tap-water was available ad libitum in water bottles.
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly using the test item as supplied.

Separate formulations were prepared for each concentration.

For dose formulations in groups 2 (100 mg/kg bw/day) and 3 (300 mg/kg/bw/day), Gel All DX 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.

For dose formulation in group 4 (1000 mg/kg bw/day), Gel All DX was weighed into a glass beaker on a tared precision balance and approximately 60% of the vehicle was added to another glass beaker. The test item was added successively to the vehicle under constant mixing. Having obtained a homogeneous mixture containing entire amount of the test item, the remaining vehicle was added.

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

STORAGE OF DOSE FORMULATIONS:
Dose formulations were stored in refrigerator (5 ± 3 °C) in glass beakers.

Based upon the results of stability analyses performed within 14-Day Dose Range-Finding Study in the Han Wistar Rat, dose formulations were stable for at least 8 days if stored in refrigerator (5 ± 3 °C).

TREATMENT:
Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Frequency of Administration: Daily, at approximately 24 hour intervals.

Dose Levels:
Group 1: 0 mg/kg/day (control group)
Group 2: 100 mg/kg/day
Group 3: 300 mg/kg/day
Group 4: 1000 mg/kg/day

Dose Volume: 5 mL/kg body weight

Dose Concentrations:
Group 1: 0 mg/mL
Group 2: 20mg/mL
Group 3: 60 mg/mL
Group 4: 200 mg/mL

Duration of Acclimatization Period: Minimum 5 days

Duration of Treatment Period:
Males: 46 days
Females: Approximately 7 weeks
Details on mating procedure:
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 on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum. No sperm or copulation plug was noted in two females, no. 84 in group 3 and no. 86 in group 4, during the first pairing period. However, as a body weight gain typical for pregnancy was observed in these females, it was considered that mating of these females was overlooked and therefore they were not paired with a second partner, but instead transferred to gestation after 13 days of the pairing period.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 post partum 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:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days at 5 ± 3 °C) samples of about 1 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. During the last week before first planned necropsies, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were delivered to the analytical laboratory immediately after collection at ambient temperature and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an UV detector. The test item was used as the analytical standard.

Duplicates were taken of all samples.

In blank sample chromatograms no peak appeared at the retention time of Gel All DX and, therefore, the absence of the test item in the vehicle control samples (olive oil) was confirmed. The Gel All DX concentrations in the dose formulations ranged from 89.5% to 102.6% with reference to the nominal and were within the accepted range of ±20%.

The homogeneous distribution of Gel All DX in the preparations was approved because single results found did not deviate more than 0.9% from the corresponding mean and met the specified acceptance criterion of ≤15%.

In addition, the test item was found to be stable in application formulations when kept 8 days in the refrigerator (5 ± 3 °C) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.

In conclusion, the results indicate the accurate preparation and storage of the test item Gel All DX in vehicle during the study.
Duration of treatment / exposure:
46 days for males and approximately 7 weeks for females
Frequency of treatment:
Daily, at approximately 24 hour intervals
Details on study schedule:
Acclimatization: 5 days minimum
First Test Item Administration: Day 1 of pre-pairing
Pre-Pairing: 14 days
Pairing: 14 days maximum
Gestation (females): Approximately 21 days
Treatment ends: On day before sacrifice (males) and on day 3 post partum (females)
Necropsy: After 46 days of treatment (males). Females and pups on Day 4 post partum.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
48 males (12 per group) and 48 females (12 per group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous 14-Day Dose Range-Finding Study in the Han Wistar Rat, using dose levels of 0, 100, 300 and 1000 mg/kg/day, resulting in a NOAEL of 1000 mg/kg/day.
Positive control:
None
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: Weekly during pre-pairing, gestation period days 0 - 7, 7 - 14 and 14 - 21 and lactation period days 1 - 4.
No food consumption was recorded during the pairing period.

BODY WEIGHTS:
Recorded daily from treatment start to day of necropsy.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight
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):
PATHOLOGY:
Males were sacrificed when they were no longer needed for the assessment of reproductive effects (after 46 days of treatment). Dams were sacrificed on day 4 post partum.

If birth did not occur, the dam was sacrificed on day 25 post coitum.

NECROPSY:
At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals, except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death.

For the parent animals, special attention was directed at the organs of the reproductive system.

Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

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, organs were weighted and preserved as listed below. Organs were trimmed from any adherent tissue, and their wet weight taken.
- Epididymides (fixed in modified Davidson's solution)
- Testes (fixed in modified Davidson's solution)

HISTOTECHNIQUE:
Initially, all organ and tissue samples listed below from control and high dose groups, any decedents and any animals failing to produce a pregnancy were processed, embedded and cut at an approximate thickness of 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis and epididymides were stained by PAS. Special stains were used at the discretion of the study pathologist.

- Epididymides (fixed in modified Davidson's solution)
- Ovaries
- Uterus (incl. oviducts, cervix and vagina)
- Prostate gland and seminal vesicles incl. coagulating glands
- Testes (fixed in modified Davidson's solution)
- All gross lesions

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 pathologist. The same applied to all occurring gross lesions and to all animals, which died spontaneously, or had to be terminated in extremis.

Qualitative assessment of the male reproductive organs was performed. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

A histopathology peer review was performed.
Postmortem examinations (offspring):
Pups were sacrified on day 4 post partum.

All pups, except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death.
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 online recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation loss.
For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis.
Offspring viability indices:
From the online recorded reproduction data, the following parameters were calculated: mean litter size, pup sex ratios and viability indices.
Mean pup weights were calculated from the individual weights both on a per group and on a per litter basis.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
VIABILITY/MORTALITY:
No test item related deaths were noted during the study.

One male given 1000 mg/kg body weight/day (no. 48) was found dead on day 12 of the after pairing period. Prior to its death a decrease in food consumption and body weight loss were noted for several days however no clinical signs or test item-related macroscopical findings were noted, which would clarify the cause of this death. Because of an isolated occurrence, it was considered to be unlikely related to toxicity of the test item.

CLINICAL SIGNS OR OBSERVATIONS:
There were no clinical signs seen in males or females at any dose level, over the course of the study that were considered to be related to administration of the test item.

Incidentally, hair loss and scabs were observed in one male (no. 24) at the dose level of 100 mg/kg bw/day and in one female (no. 84) at the dose level of 300 mg/kg bw/day. Hair loss was also noted in one female (no. 96) at the dose level of 1000 mg/kg bw/day.

Further, one female (no. 74) at the dose level of 300 mg/kg bw/day was observed with ruffled fur and one further female (no. 93) at the dose level of 1000 mg/kg bw/day was observed with ruffled fur and decreased activity on day 22 post coitum. Female no. 93 had ruffled fur also during lactation and it neglected its litter.

For both females, the onset of the clinical sign corresponded with the day of parturition. Therefore, as these findings were isolated incidents and were distributed across the dose levels, it was considered that they were attributable to parturition and not to administration of the test item.

FOOD CONSUMPTION OF MALES:
Pre-pairing and Afer Pairing Periods:
There was no effect of the test item on the group mean amount of food consumed by males at any dose level over the course of this study, when compared to control animals.

Mean food consumption at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day was respectively: 23.3, 23.4, 22.8 and 24.2 g/animal/day during pre-pairing and 19.1, 20.2, 19.2 and 20.2 g/animal/day during after pairing periods.

At the dose level of 1000 mg/kg bw/day, statistically significantly higher food consumption was noted from day 15 to 21 of after pairing period. Mean food consumption during this period was 20.8 g/animal/day at this dose level compared to 17.9 g/animal/day in the control group. A slight decrease of food consumption in the control group contributed to this effect and therefore it was considered not to be test item-related but a result of biological variability.

FOOD CONSUMPTION OF FEMALES:
Pre-pairing, Gestation and Lactation Periods:
There was no effect of the test item on the group mean amount of food consumed by females at any dose level over the course of this study, when compared to control animals.

Mean food consumption at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day was respectively: 15.6, 15.2, 15.4 and 15.9 g/animal/day during pre-pairing, 20.8, 19.9, 20.3 and 21.2 g/animal/day during gestation and 26.4, 23.1, 25.0 and 23.5 g/animal/day during lactation periods.

BODY WEIGHTS OF MALES:
Pre-Pairing, Pairing and After Pairing Periods:
Group mean body weight and body weight gain for males, were considered to be unaffected by administration of the test item over the duration of the study, when compared to control animals.

Mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day was respectively: 12%, 14%, 13% and 14% during pre-pairing, 4%, 6%, 6% and 7% during pairing and 6%, 7%, 5% and 6% during after pairing periods.

Statistically significantly higher body weight gain was occasionally observed in all treated groups during the pairing period. The changes were not clearly dose dependent and therefore this observation was considered not to be related to the treatment with the test item but probably be due to incidentally lower body weight gain in the control group.

Statistically significantly lower body weight gain was noted at the dose level of 1000 mg/kg bw/day from day 11 to 14 of after pairing period. The differences to the respective values in the control group were only minor; 2% were noted at the high-dose level and 4% in the control group. During the same period, absolute body weights at the high-dose level were slightly higher than body weights in the control group and therefore this difference was considered to be a result of biological variability and not test item-related.

BODY WEIGHTS OF FEMALES:
Pre-Pairing, Pairing, Gestation and Lactation Periods:
Group mean body weight and body weight gain for females, were considered to be unaffected by administration of the test item over the duration of the study, when compared to control animals.

Mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day was respectively: 8%, 9%, 9%, 8% during pre-pairing, 57%, 58%, 61% and 60% during gestation and 3%, 3%, 2%, 4% during lactation periods.

MATING PERFORMANCE AND FERTILITY:
No effect on mating performance or fertility was observed at any dose level.

With exception for female no. 84 at the dose level of 300 mg/kg body weight/day and female no. 86 at the dose level of 1000 mg/kg body weight/day, all females mated during the first six days of the pairing period. The precoital time was not affected by the treatment with the test item. Mean (median) precoital times calculated for the first pairing period were 3.3 (3), 3.2 (3), 2.6 (3) and 2.9 (3) days in order of ascending dose levels.

Mating of female nos. 84 and 86 was not observed during the first pairing period. Significant body weight gain was recorded at the end of this period in both females. It was considered that the body weight gain indicated pregnancy after the mating was overlooked. For this reason, females 84 and 86 were not mated with a second male partner. During necropsy it was verified that these females were not pregnant.

Two females in the control group were not pregnant (nos. 50 and 59). Fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 83.3% in the control group and 100% in the remaining groups.

No birth was recorded for one female at the dose level of 1000 mg/kg bw/day (no. 95). At termination on day 25 post coitum, only resorptions/implantation sites were found in this female, proving that she had successfully mated. All remaining females gave birth to living pups. Gestation index (number of females with living pups as a percentage of females pregnant) was 100% in the control group and at the dose levels of 100 and 300 mg/kg bw/day and 90.9% at the dose level of 1000 mg/kg bw/day.

DURATION OF GESTATION:
No effects on duration of gestation were observed at any dose level. Mean duration of gestation was 21.6, 21.3, 21.5 and 21.4 days, for groups given 0, 100, 300 or 1000 mg/kg bw/day, respectively.

CORPORA LUTEA COUNT:
No effects on corpora lutea count were observed at any dose level. Mean number of corpora lutea per dam was 16.4, 15.8, 16.6 and 16.8 in order of ascending dose levels.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS:
At the dose levels of 100 and 300 or 1000 mg/kg bw/day, no effects on implantation rate or post-implantation loss were noted. The overall number of implantations per dam was 12.5, 13.2, 13.7 and 13.4 with a corresponding mean number of post-implantation losses per dam of 1.3, 1.3, 1.5 and 1.0 at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively.


PATHOLOGY (PARENT ANIMALS):
ORGAN WEIGHTS:
No changes in absolute testes and epididymides weights or weights of these organs relative to body weight at any dose level.

MACROSCOPIC FINDINGS:
No test item-related observations were recorded during necropsy at any dose level.

In males, alopecia and eschar were noted in one male (no. 24) in the low-dose group, and advance autolysis was recorded for one male which was found dead (no. 48) at the high-dose level.
In females, shortened left horn was noted in one female (no. 67) at the low-dose level, hardened inguinal mammary gland was noted in one female (no. 82) in the mid-dose group and fetal resorptions were found in one female (no. 95) at the high-dose level.

All these findings were typical of this strain and age of rat and were considered to be incidental.

MICROSCOPIC FINDINGS:
No test item-related observations were recorded in this study.

All findings observed in this study were considered to be of spontaneous background encountered in this strain and type of study.
Key result
Dose descriptor:
NOEL
Remarks:
reproduction/developmental toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect on mating performance or fertility was observed at any dose level.
Dose descriptor:
NOEL
Remarks:
general toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item related effects on any parameter measured, at any dose level.
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
OBSERVATIONS (LITTER DATA - F1 PUPS):

LITTER SIZE AT FIRST LITTER CHECK:
No effects on litter size were observed at any dose level. Mean number of pups per dam was 11.2, 11.9, 12.3 and 12.4 in order of ascending dose levels. Birth index (number of pups born alive as a percentage of implantations) was 89.6%, 90.5%, 89.4% and 92.5% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM:
At the dose level of 1000 mg/kg body weight/day, statistically significantly increased post natal loss was recorded; 13 pups at this dose level were missing or found dead on day 2 or 3 of the lactation period whereas no postnatal loss was recorded in the control group. Viability index (number of pups alive on day 4 post partum as a percentage of pups born alive) was 100.0%, 97.2%, 99.3% and 89.5% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Reduction of viability index at the high-dose level was statistically significant.

All pups lost during the lactation at the high dose level were confined to the litter of one female (no. 93). As no pup deaths were recorded in any other litter at this dose level the loss of litter no. 93 was considered to be incidental and not related to treatment with the test item.


EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION:
No test-item related findings were noted in pups at first litter check or during lactation at any dose level.

During lactation, scab was noted for two pups from two litters (nos. 69 and 71) at the dose level of 100 mg/kg bw/day. In the neglected litter no. 93 at the dose level 1000 mg/kg bw/day, two pups were found with weakened condition and cold to touch on day 2 post partum. These pups were missing on day 3 post partum.

SEX RATIOS:
Pups sex ratio was not affected by exposure to the test item at any dose level.

At first litter check, percentages of male pups were 50%, 57%, 44% and 48%, in order of ascending dose level.

BODY WEIGHTS TO DAY 4 POST PARTUM:
No effects on pup body weights or body weight gain were noted at any dose level.

Mean body weights of pups per group on day 1 post partum was 6.43, 6.11, 6.22 and 6.07 g whereas mean body weight gain during lactation was 47.70%, 50.36%, 48.25% and 47.31%, both in order of ascending dose levels

MACROSCOPIC FINDINGS:
No test item-related findings were noted in pups at necropsy at any dose level.

Scab recorded for pup in litter no. 92 at the dose level of 100 mg/kg bw/day during lactation was confirmed during necropsy as sore. No further findings were recorded at any dose level.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect on mating performance or fertility was observed at any dose level.
Reproductive effects observed:
not specified
Conclusions:
The NOEL (No Observed Effect Level) for both general toxicity in males and females for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.
Executive summary:

General

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

 

The test item was administered once daily orally (by gavage) throughout pre-pairing, pairing and after pairing periods for a total of 46 days in males and pre-pairing, pairing, gestation and lactation periods for at least 40 days in females, up to the day before scheduled necropsy.

 

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 (olive oil).

Parent Animals

Mortality/Viability

No test item related deaths were noted during the study.

 

One male given 1000 mg/kg body weight/day was found dead on day 12 of the after pairing period. The cause of this death was unclear and, due to an isolated occurrence, unlikely to be related to toxicity of the test item.

Clinical Signs

There were no clinical signs seen in males or females at any dose level, over the course of the study that were considered to be related to the administration of the test item.

Food Consumption

There was no effect of the test test item on the group mean amount of food consumed by males or females at any dose level over the course of this study, when compared to control animals.

Body Weights

Group mean body weight and body weight gain for males and females were considered to be unaffected by administration of the test item over the duration of the study.

Reproduction and Breeding Data

Fertility, time course of mating, duration of gestation, corpora lutea count, number of implantation sites, post-implantation loss, liter size and post natal loss were unaffected by administration of the test item at any dose level.

Organs Weights

No changes in absolute testes and epididymides weights or weights of these organs relative to body weight were noted at any dose level.

Macroscopic/Microscopic Findings

No test item-related macroscopical or microscopical findings were recorded at any dose level.

Litter Data - F1 Pups

Findings at First Litter Check and During Lactation

No test-item related findings were noted in pups at first litter check or during lactation at any dose level.

 

Pups sex ratio was not affected by exposure to the test item at any dose level.

 

Pup Weights to Day 4 Post Partum

No effects on pup body weights or body weight gain were noted at any dose level.

Macroscopic Findings

No test item-related findings were noted in pups at necropsy at any dose level.

Conclusion

Based on these results, NOEL (No Observed Effect Level) for both general toxicity in males and females for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adapted 27th July 1995
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
Animals: Rat, RccHanTM: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V., Netherlands
Number of Animals: 48 males: 12 per group
48 females: 12 per group
Age (at Start of Treatment): Approximately 11 weeks
Body Weight Range (at Start of Treatment): Males 329 to 382 g
Females 185 to 236 g
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. 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.

HUSBANDRY:
Conditions:
Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 18 ± 3 °C from 13-Jun-2013 to 26-Jun-2013 and 22 ± 3 °C from 27-Jun-20113 onwards; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, which was considered not to have any influence on the study. The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle

Accommodation:
Individually in Makrolon type-3 cages with wire mesh tops and sterilized stan¬dard softwood bedding with paper enrichment. 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 Harlan Teklad 2018C rodent maintenance diet was available ad libitum.

Water:
Community tap-water was available ad libitum in water bottles.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly using the test item as supplied.

Separate formulations were prepared for each concentration.

For dose formulations in groups 2 (100 mg/kg bw/day) and 3 (300 mg/kg/bw/day), Gel All DX 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.

For dose formulation in group 4 (1000 mg/kg bw/day), Gel All DX was weighed into a glass beaker on a tared precision balance and approximately 60% of the vehicle was added to another glass beaker. The test item was added successively to the vehicle under constant mixing. Having obtained a homogeneous mixture containing entire amount of the test item, the remaining vehicle was added.

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

STORAGE OF DOSE FORMULATIONS:
Dose formulations were stored in refrigerator (5 ± 3 °C) in glass beakers.

Based upon the results of stability analyses performed within 14-Day Dose Range-Finding Study in the Han Wistar Rat, dose formulations were stable for at least 8 days if stored in refrigerator (5 ± 3 °C).

TREATMENT:
Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Frequency of Administration: Daily, at approximately 24 hour intervals.

Dose Levels:
Group 1: 0 mg/kg/day (control group)
Group 2: 100 mg/kg/day
Group 3: 300 mg/kg/day
Group 4: 1000 mg/kg/day

Dose Volume: 5 mL/kg body weight

Dose Concentrations:
Group 1: 0 mg/mL
Group 2: 20mg/mL
Group 3: 60 mg/mL
Group 4: 200 mg/mL

Duration of Acclimatization Period: Minimum 5 days

Duration of Treatment Period:
Males: 46 days
Females: Approximately 7 weeks
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days at 5 ± 3 °C) samples of about 1 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. During the last week before first planned necropsies, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were delivered to the analytical laboratory immediately after collection at ambient temperature and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an UV detector . The test item was used as the analytical standard.

Duplicates were taken of all samples.

In blank sample chromatograms no peak appeared at the retention time of Gel All DX and, therefore, the absence of the test item in the vehicle control samples (olive oil) was confirmed. The Gel All DX concentrations in the dose formulations ranged from 89.5% to 102.6% with reference to the nominal and were within the accepted range of ±20%.

The homogeneous distribution of Gel All DX in the preparations was approved because single results found did not deviate more than 0.9% from the corresponding mean and met the specified acceptance criterion of ≤15%.

In addition, the test item was found to be stable in application formulations when kept 8 days in the refrigerator (5 ± 3 °C) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.

In conclusion, the results indicate the accurate preparation and storage of the test item Gel All DX in vehicle during the study.
Details on mating procedure:
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 on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum. No sperm or copulation plug was noted in two females, no. 84 in group 3 and no. 86 in group 4, during the first pairing period. However, as a body weight gain typical for pregnancy was observed in these females, it was considered that mating of these females was overlooked and therefore they were not paired with a second partner, but instead transferred to gestation after 13 days of the pairing period.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 post partum was designated as the day on which a female had delivered all her pups.
Duration of treatment / exposure:
46 days for males and approximately 7 weeks for females
Frequency of treatment:
Daily, at approximately 24 hour intervals
Duration of test:
See duration of treatment
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
48 males (12 per group) and 48 females (12 per group)
Control animals:
yes, concurrent vehicle
Details on study design:
Study schedule:
Acclimatization: 5 days minimum
First Test Item Administration: Day 1 of pre-pairing
Pre-Pairing: 14 days
Pairing: 14 days maximum
Gestation (females): Approximately 21 days
Treatment ends: On day before sacrifice (males) and on day 3 post partum (females)
Necropsy: After 46 days of treatment (males). Females and pups on Day 4 post partum.

Examinations

Maternal 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 pair-ing periods.
Females: Weekly during pre-pairing, gestation period days 0 - 7, 7 - 14 and 14 - 21 and lactation period days 1 - 4.
No food consumption was recorded during the pairing period.

BODY WEIGHTS:
Recorded daily from treatment start to day of necropsy.

PATHOLOGY:
Males were sacrificed when they were no longer needed for the assessment of reproductive effects (after 46 days of treatment). Dams were sacrificed on day 4 post partum.

If birth did not occur, the dam was sacrificed on day 25 post coitum.

NECROPSY:
At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals, except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death.

For the parent animals, special attention was directed at the organs of the reproductive system.

Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

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, organs were weighted and preserved as listed below. Organs were trimmed from any adherent tissue, and their wet weight taken.
- Epididymides (fixed in modified Davidson's solution)
- Testes (fixed in modified Davidson's solution)

HISTOTECHNIQUE:
Initially, all organ and tissue samples listed below from control and high dose groups, any decedents and any animals failing to produce a pregnancy were processed, embedded and cut at an approximate thickness of 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis and epididymides were stained by PAS. Special stains were used at the discretion of the study pathologist.

- Epididymides (fixed in modified Davidson's solution)
- Ovaries
- Uterus (incl. oviducts, cervix and vagina)
- Prostate gland and seminal vesicles incl. coagulating glands
- Testes (fixed in modified Davidson's solution)
- All gross lesions

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 pathologist. The same applied to all occurring gross lesions and to all animals, which died spontaneously, or had to be terminated in extremis.

Qualitative assessment of the male reproductive organs was performed. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

A histopathology peer review was performed.
Fetal examinations:
See litter data.
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.
Indices:
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation loss.
For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis.
From the on-line recorded reproduction data, the following parameters were calculated: mean litter size, pup sex ratios and viability indices.
Mean pup weights were calculated from the individual weights both on a per group and on a per litter basis.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
VIABILITY/MORTALITY:
No test item related deaths were noted during the study.

One male given 1000 mg/kg body weight/day (no. 48) was found dead on day 12 of the after pairing period. Prior to its death a decrease in food consumption and body weight loss were noted for several days however no clinical signs or test item-related macroscopical findings were noted, which would clarify the cause of this death. Because of an isolated occurrence, it was considered to be unlikely related to toxicity of the test item.

CLINICAL SIGNS OR OBSERVATIONS:
There were no clinical signs seen in males or females at any dose level, over the course of the study that were considered to be related to administration of the test item.

Incidentally, hair loss and scabs were observed in one male (no. 24) at the dose level of 100 mg/kg bw/day and in one female (no. 84) at the dose level of 300 mg/kg bw/day. Hair loss was also noted in one female (no. 96) at the dose level of 1000 mg/kg bw/day.

Further, one female (no. 74) at the dose level of 300 mg/kg bw/day was observed with ruffled fur and one further female (no. 93) at the dose level of 1000 mg/kg bw/day was observed with ruffled fur and decreased activity on day 22 post coitum. Female no. 93 had ruffled fur also during lactation and it neglected its litter.

For both females, the onset of the clinical sign corresponded with the day of parturition. Therefore, as these findings were isolated incidents and were distributed across the dose levels, it was considered that they were attributable to parturition and not to administration of the test item.

FOOD CONSUMPTION OF MALES:
Pre-pairing and Afer Pairing Periods:
There was no effect of the test item on the group mean amount of food consumed by males at any dose level over the course of this study, when compared to control animals.

Mean food consumption at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day was respectively: 23.3, 23.4, 22.8 and 24.2 g/animal/day during pre-pairing and 19.1, 20.2, 19.2 and 20.2 g/animal/day during after pairing periods.

At the dose level of 1000 mg/kg bw/day, statistically significantly higher food consumption was noted from day 15 to 21 of after pairing period. Mean food consumption during this period was 20.8 g/animal/day at this dose level compared to 17.9 g/animal/day in the control group. A slight decrease of food consumption in the control group contributed to this effect and therefore it was considered not to be test item-related but a result of biological variability.

FOOD CONSUMPTION OF FEMALES:
Pre-pairing, Gestation and Lactation Periods:
There was no effect of the test item on the group mean amount of food consumed by females at any dose level over the course of this study, when compared to control animals.

Mean food consumption at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day was respectively: 15.6, 15.2, 15.4 and 15.9 g/animal/day during pre-pairing, 20.8, 19.9, 20.3 and 21.2 g/animal/day during gestation and 26.4, 23.1, 25.0 and 23.5 g/animal/day during lactation periods.

BODY WEIGHTS OF MALES:
Pre-Pairing, Pairing and After Pairing Periods:
Group mean body weight and body weight gain for males, were considered to be unaffected by administration of the test item over the duration of the study, when compared to control animals.

Mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day was respectively: 12%, 14%, 13% and 14% during pre-pairing, 4%, 6%, 6% and 7% during pairing and 6%, 7%, 5% and 6% during after pairing periods.

Statistically significantly higher body weight gain was occasionally observed in all treated groups during the pairing period. The changes were not clearly dose dependent and therefore this observation was considered not to be related to the treatment with the test item but probably be due to incidentally lower body weight gain in the control group.

Statistically significantly lower body weight gain was noted at the dose level of 1000 mg/kg bw/day from day 11 to 14 of after pairing period. The differences to the respective values in the control group were only minor; 2% were noted at the high-dose level and 4% in the control group. During the same period, absolute body weights at the high-dose level were slightly higher than body weights in the control group and therefore this difference was considered to be a result of biological variability and not test item-related.

BODY WEIGHTS OF FEMALES:
Pre-Pairing, Pairing, Gestation and Lactation Periods:
Group mean body weight and body weight gain for females, were considered to be unaffected by administration of the test item over the duration of the study, when compared to control animals.

Mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day was respectively: 8%, 9%, 9%, 8% during pre-pairing, 57%, 58%, 61% and 60% during gestation and 3%, 3%, 2%, 4% during lactation periods.

MATING PERFORMANCE AND FERTILITY:
No effect on mating performance or fertility was observed at any dose level.

With exception for female no. 84 at the dose level of 300 mg/kg body weight/day and female no. 86 at the dose level of 1000 mg/kg body weight/day, all females mated during the first six days of the pairing period. The precoital time was not affected by the treatment with the test item. Mean (median) precoital times calculated for the first pairing period were 3.3 (3), 3.2 (3), 2.6 (3) and 2.9 (3) days in order of ascending dose levels.

Mating of female nos. 84 and 86 was not observed during the first pairing period. Significant body weight gain was recorded at the end of this period in both females. It was considered that the body weight gain indicated pregnancy after the mating was overlooked. For this reason, females 84 and 86 were not mated with a second male partner. During necropsy it was verified that these females were not pregnant.

Two females in the control group were not pregnant (nos. 50 and 59). Fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 83.3% in the control group and 100% in the remaining groups.

No birth was recorded for one female at the dose level of 1000 mg/kg bw/day (no. 95). At termination on day 25 post coitum, only resorptions/implantation sites were found in this female, proving that she had successfully mated. All remaining females gave birth to living pups. Gestation index (number of females with living pups as a percentage of females pregnant) was 100% in the control group and at the dose levels of 100 and 300 mg/kg bw/day and 90.9% at the dose level of 1000 mg/kg bw/day.

DURATION OF GESTATION:
No effects on duration of gestation were observed at any dose level. Mean duration of gestation was 21.6, 21.3, 21.5 and 21.4 days, for groups given 0, 100, 300 or 1000 mg/kg bw/day, respectively.

CORPORA LUTEA COUNT:
No effects on corpora lutea count were observed at any dose level. Mean number of corpora lutea per dam was 16.4, 15.8, 16.6 and 16.8 in order of ascending dose levels.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS:
At the dose levels of 100 and 300 or 1000 mg/kg bw/day, no effects on implantation rate or post-implantation loss were noted. The overall number of implantations per dam was 12.5, 13.2, 13.7 and 13.4 with a corresponding mean number of post-implantation losses per dam of 1.3, 1.3, 1.5 and 1.0 at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively.


PATHOLOGY (PARENT ANIMALS):
ORGAN WEIGHTS:
No changes in absolute testes and epididymides weights or weights of these organs relative to body weight at any dose level.

MACROSCOPIC FINDINGS:
No test item-related observations were recorded during necropsy at any dose level.

In males, alopecia and eschar were noted in one male (no. 24) in the low-dose group, and advance autolysis was recorded for one male which was found dead (no. 48) at the high-dose level.
In females, shortened left horn was noted in one female (no. 67) at the low-dose level, hardened inguinal mammary gland was noted in one female (no. 82) in the mid-dose group and fetal resorptions were found in one female (no. 95) at the high-dose level.

All these findings were typical of this strain and age of rat and were considered to be incidental.

MICROSCOPIC FINDINGS:
No test item-related observations were recorded in this study.

All findings observed in this study were considered to be of spontaneous background encountered in this strain and type of study.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: other:

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOEL (No Observed Effect Level) for both general toxicity in males and females for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.
Executive summary:

General

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

 

The test item was administered once daily orally (by gavage) throughout pre-pairing, pairing and after pairing periods for a total of 46 days in males and pre-pairing, pairing, gestation and lactation periods for at least 40 days in females, up to the day before scheduled necropsy.

 

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 (olive oil).

Parent Animals

Mortality/Viability

No test item related deaths were noted during the study.

 

One male given 1000 mg/kg body weight/day was found dead on day 12 of the after pairing period. The cause of this death was unclear and, due to an isolated occurrence, unlikely to be related to toxicity of the test item.

Clinical Signs

There were no clinical signs seen in males or females at any dose level, over the course of the study that were considered to be related to the administration of the test item.

Food Consumption

There was no effect of the test test item on the group mean amount of food consumed by males or females at any dose level over the course of this study, when compared to control animals.

Body Weights

Group mean body weight and body weight gain for males and females were considered to be unaffected by administration of the test item over the duration of the study.

Reproduction and Breeding Data

Fertility, time course of mating, duration of gestation, corpora lutea count, number of implantation sites, post-implantation loss, liter size and post natal loss were unaffected by administration of the test item at any dose level.

Organs Weights

No changes in absolute testes and epididymides weights or weights of these organs relative to body weight were noted at any dose level.

Macroscopic/Microscopic Findings

No test item-related macroscopical or microscopical findings were recorded at any dose level.

Litter Data - F1 Pups

Findings at First Litter Check and During Lactation

No test-item related findings were noted in pups at first litter check or during lactation at any dose level.

 

Pups sex ratio was not affected by exposure to the test item at any dose level.

 

Pup Weights to Day 4 Post Partum

No effects on pup body weights or body weight gain were noted at any dose level.

Macroscopic Findings

No test item-related findings were noted in pups at necropsy at any dose level.

Conclusion

Based on these results, NOEL (No Observed Effect Level) for both general toxicity in males and females for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.


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