Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2010-02-18 to 2011-02-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted GLP guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test), July 2000
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Han Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS: Rat, RccHanTM: WIST(SPF)
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 295 to 343 g; Females: 172 to 213 g
- Fasting period before study: no
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 60/09).
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles
- Acclimation period: 8 days

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

IN-LIFE DATES: From: 18 February 2010 To: 22 April 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly.The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no. : 049103168 (expiry date: 12-Oct-2014)
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days maximum
- Proof of pregnancy: the daily vaginal smear was sperm positive, or a copulation plug was observed. Referred to as day 0 of pregnancy.
- Any other deviations from standard protocol: Due to the mortality during the pre-pairing period the number of males was lower than the number of females at the start of the pairing period, the leftover females were paired with a male of the same group which had already mated successfully. Female no. 100 which replaced the death female no. 62 was paired with male no. 95 which replaced one of the dead males. Because upon completion of the pre-pairing period the number of additional males was higher than the number of additional females, the remaining males (nos. 93, 94, 96, 97 and 98) were not paired.
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 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 in which all dose formulations were prepared, 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 R. Geissbühler (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.
The samples were analysed by GC coupled to an FI detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard.
Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.
Duration of treatment / exposure:
Males: Minimum 4 weeks
Females: Approximately 7 weeks
Frequency of treatment:
Once daily
Details on study schedule:
First Test Item Administration: Day 1 of pre-pairing
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
30 mg/kg bw/d
Basis:
actual ingested
Group 2
Remarks:
Doses / Concentrations:
60 mg/kg bw/d
Basis:
actual ingested
Group 3
Remarks:
Doses / Concentrations:
90 mg/kg bw/d
Basis:
actual ingested
Group 4
No. of animals per sex per dose:
46 males: 10 per group (in groups 1 and 2), 13 per group (in groups 3 and 4)
41 females: 10 per group (in groups 1,2 and 4); 11 per group (in group 3)
Initially 41 males and 41 females were obtained from the breeder and 10 animals per group were allocated. After death of several animals, additional 6 males and 5 females were obtained and allocated to the main groups.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Han Wistar Rats, Harlan Laboratories Study C48852, using dose levels of 5, 15, 50, 120 and 150 mg/kg/day.
- Rationale for animal assignment (if not random): random

Examinations

Parental animals: Observations and examinations:
VIABILITY/MORTALITY: Yes
- Time schedule: Twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily (once daily, during acclimatization and up to day of necropsy).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed in main groups. Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage.
- Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also reported.

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

FOOD CONSUMPTION : Yes
- Time schedule:
Males: Weekly during pre-pairing and after pairing periods
Females: Pre-pairing period days 1 - 8 and 8 - 14; 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.

WATER CONSUMPTION AND COMPOUND INTAKE: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of the scheduled necropsy for males, on day 5 post-partum for lactating females .
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: 5 males and 5 females per group
- Parameters checked in table 7.8.1/2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of the scheduled necropsy for males, on day 5 post-partum for lactating females.
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: 5 males and 5 females per group
- Parameters checked in table 7.8.1/2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At one time during the study (males shortly before the scheduled sacrifice and females on day 3 post-partum (separation time from the pups not longer than 30 - 40 minutes)) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration.
- Battery of functions tested:
• Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
• Hand-held observations: muscle tone, constitution, skin, pupil size, palpebral closure,
lacrimation, salivation, reaction to handling and general abnormalities.
• Open field observations: level of ambulatory activity including rearing (one minute
evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation,
behaviour, hair coat, respiration, quantity of faeces-balls and urine.
• Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch,
responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
• Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal
temperature
• Locomotor activity

OTHER: Additionally females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.
Oestrous cyclicity (parental animals):
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.
Sperm parameters (parental animals):
Parameters examined in all male parental generations: testis weight, epididymis weight.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring: litter size, live births, still births, presence of gross anomalies, sex ration, weight on days 0 (if possible), and 4 post-partum.

GROSS EXAMINATION OF DEAD PUPS:
yes, Pups sacrificed before scheduled necropsy or found dead were, where condition of the tissue allowed, fixed in ethanol as contingency against possible detailed examination.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after treatment of at least 28 days, when no longer needed for assessment of reproductive effects.
- Maternal animals: All surviving animals were sacrificed on day 5 post-partum (pups on day 4 post-partum). If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 7.8.1/3 were prepared for microscopic examination and weighed, respectively.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. If test item-related morphologic changes were detected in organs of any high-dose animal, those same organs from the mid- and low-dose group were examined to establish a no-effect level, if possible.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.
Postmortem examinations (offspring):
SACRIFICE
- Pups were sacrificed on day 4 post-partum
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
1. External and visceral examination of pups/ Approximately one half of the pups from each litter was allocated to this procedure;
2. Skeletal examination of pups/ The other half of the pups from each litter was allocated to this procedure.
Statistics:
The following statistical methods were used to analyze functional observational battery, food consumption, body weights, reproduction data, clinical laboratory investigations and organ weight and ratios:
• 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:
Precoital time, fertility index, conception rate, gestation index, duration of Gestation, Corpora Lutea Count, Implantation Rate and Post-implantation Loss
Offspring viability indices:
Viability index

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
at all dose levels: bedding in mouth, breathing noises and salivation
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced body weight and body weight gain in mid- and high-dose males, reduced food consumption in high-dose males
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
reduced body weight and body weight gain in mid- and high-dose males, reduced food consumption in high-dose males
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
results of test item irritant properties
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
results of test item irritant properties
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Mating performance was considered to be affected by the treatment with the test item at the dose level of 90 mg/kg bw/day.

Details on results (P0)

MORTALITY (PARENTAL ANIMALS)
During the treatment nine males and one female were found dead before scheduled necropsy: four males at the dose level of 90 mg/kg bw/day, four males and one female at the dose level of 60 mg/kg bw/day and one male at the dose level of 30 mg/kg bw/day. All these deaths were considered to be related to the treatment with the test item. During histopathological examination necrosis of trachea was identified as a cause of all preterm deaths. Effects on animal airways and animal deaths during the study were considered to be a result of corrosive and/or irritative properties of the test item.

CLINICAL SIGNS (PARENTAL ANIMALS)
At the dose level of 90 mg/kg bw/day bedding in mouth was observed in up to 100% of males and females, breathing noises were observed in up to 60% of males and up to 30% of females, one female had dyspnea for 1 day, salivation was observed in four males.
At the dose level of 60 mg/kg bw/day bedding in mouth was observed in up to 40% of males and up to 100% of females, breathing noises were observed in up to 40% of males and 30% of females, salivation was observed in three males and 2 females.
At the dose level of 30 mg/kg bw/day breathing noises were observed in one male on day 11 of the pre-pairing period.
Further findings: ruffled fur observed in one male and one female at the dose level of 90 mg/kg bw/day, soft faeces observed in another male at the dose level of 90 mg/kg bw/day and ruffled fur and diarrhea in one male at the dose level of 60 mg/kg bw/day were considered to be related to the treatment with the test item and to be signs of a bad condition of animals.

BODY WEIGHT (PARENTAL ANIMALS)
- Males
At the dose level of 90 mg/kg bw/day lower body weights were observed during the pre-pairing and pairing periods. Compared to the control values, reduction of body weights was statistically significant from day 5 of the pre-pairing period onwards. During the pre-pairing period reduction of mean body weight gain was observed. This reduction was statistically significant from day 3 to 14 of this period. In eight males body weight loss was observed for several days. Four of these males were found dead. The effects on body weights and body weight gain at this dose level were considered to be adverse. At the dose level of 60 mg/kg bw/day if compared to the control values lower mean body weights were observed from day 2 of the pre-pairing period onwards. This difference was statistically significant on days 1, 5, 6, 14 and 15 of the pairing period. Mean body weight gain was reduced during the pre-pairing period; this reduction was statistically significant on days 3 and 12 to 14 of the pre-pairing period. No apparent reduction of mean body weight gain was noted during the pairing period. In four males a body weight loss was observed for several days. All these males were found dead before scheduled termination. The effects on body weights and body weight gain at this dose level were considered to be adverse. No effects on body weights and body weight gain were observed at the dose level of 30 mg/kg bw/day. Difference in mean body weight gain at the dose levels of 0, 30, 60 and 90 were respectively +10%, +9%, +6% and +2% during the pre-pairing period and +4%, +3%, +1% and +1% during the pairing period (percentages refer to the body weight gain within the period).
- Females
No effects on body weights or body weight gain of females which were considered to be test item-related were observed at any dose level

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Males
At the dose level of 90 mg/kg bw/day mean food consumption was statistically significantly decreased during the whole pre-pairing period. Mean food consumption over this period was 15.9 g/animal/day compared to 20.7 g/animal/day in the control group (-23.2%).
At the dose level of 60 mg/kg bw/day mean food consumption was lower than the respective control value. Mean food consumption over pre-pairing period was 18.6 g/animal/day. Reduction of food consumption at this dose level was not statistically significant (-10.1%, not statistically significatif).
No effects on food consumption which were considered to be test item-related were observed at the dose level of 30 mg/kg bw/day. Mean food consumption over pre-pairing period at this dose level was 20.1 g/animal/day (-2.9%, not statistically significatif).
- Females
Treatment with the test item resulted in a slight reduction of food consumption in females at the dose level of 90 mg/kg bw/day. This reduction was considered not to be adverse.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) See Table 7.8.1/4 & 7.8.1/5
Mating performance was considered to be affected by the treatment with the test item at the dose level of 90 mg/kg bw/day.
At this dose level prolongation of precoital time was observed. Values of mean (median) precoital time were 5.3 (4) days at the high dose level compared to 3.5 (2) days in the control group. The prolongation was mostly due to the longer precoital time of three males/females. No macroscopical or histopathological changes in the reproductive organs of these males and females were found. Bad condition of the males was considered to be a reason for the effect on mating performance. Because no effect on the number of offspring was observed at this dose level, the prolongation of precoital time was considered not to be adverse.
With exception for one female at the dose level of 30 mg/kg bw/day and two females at the dose level of 90 mg/kg bw/day, all females were pregnant. Consequently, 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 100%, 90%, 100% and 80% at the dose levels of 0, 30, 60 and 90 mg/kg bw/day, respectively.
No macroscopical or histopathological changes in the reproductive organs of the infertile males and females were found. During necropsy cervix and uterine horn dilation and both organs containing fluid were found in one female. This finding was considered to be related to the estrus cycle and not a signs of any pathology. Lack of pregnancy in one female at the dose level of 30 mg/kg bw/day and two females at the dose level of 90 mg/kg bw/day was considered to be incidental.
Gestation indexes (number of females with living pups as a percentage of females pregnant) were 100%, 100%, 90% and 100% at the dose levels of 0, 30, 60 and 90 mg/kg bw/day, respectively. At the dose level of 60 mg/kg bw/day one female did not give birth. During necropsy of this female only implantation sites were identified. No macroscopical or histopathological changes in the reproductive organs of these male and female were found. Loss of the litter in this female was considered to be incidental.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No effects on organ and organ/body weights ratios which were considered to be a primary effect related to the treatment with the test item were found at any dose level in males or in females.
In the absence of any histopathological findings, effects on heart and kidneys weights observed in the high-dose males were considered to be secondary effect related to the reduced body weights of males.

GROSS PATHOLOGY (PARENTAL ANIMALS) See attachment: C48863_Macroscopical findings
- Males
Changes which were considered to be related to the treatment with the test item were found in the digestive tract at the dose level of 90 mg/kg bw/day and in the respiratory tract at the dose levels of 90 and 60 mg/kg bw/day. At the dose level of 90 mg/kg bw/day isolated focus was found in stomach of one male and isolated crateriform retraction was found in forestomach of another male. In one male at the dose level of 90 mg/kg bw/day noncollapsed lungs and in two males at the dose level of 60 mg/kg bw/day incompletely collapsed lungs were found. Findings in the stomach were considered to be a result of the irritative or corrosive properties of the test item. Also effects in the lungs were attributed to these properties. Breathing noises observed in several males at all dose levels implied a penetration of the test item into the airways and this was further supported by the findings during the histopathological analyses. The irritative changes in the lung caused by the test item were considered to be a reason for the noncollapsed
or incompletely collapsed lungs. Because of the nature and lack of a dose level dependency following findings were considered not to be test item-related: red foci on thymus (in 5 males), renal pelvic dilation (in 2 males), deformation of spleen (in 1 male) and thickened thymus.
- Females
No macroscopical findings which were considered to be test item-related were found in females at any dose level.
Cervix and uterine horn dilation and both organs containing fluid found in 1 female were considered to most probably be related to the oestrus cycle.
Because of isolated occurrence papillary process and diaphragmatic hernia of liver found in 1 female was considered to be incidental.
Dark red discoloration of the lungs and stomach, jejunum and ileum distended with gas in 1 female which was found dead were considered to possibly arise peri/post-mortem.

HISTOPATHOLOGY (PARENTAL ANIMALS) See Table 7.8.1/6
Histopathological changes which were considered to be test item-related were found in the stomach of males treated with the test item at the dose levels of 30, 60 and 90 mg/kg bw/day and females at the dose level of 90 mg/kg bw/day as well as in the lung and trachea of males at the dose levels of 30, 60 and 90 mg/kg bw/day and in one female at the dose level of 90 mg/kg bw/day. All these changes were considered to result from irritative properties of the test item but not from a systemic toxicity. A histopathological examination of the reproductive organs in males and ovaries in females did not reveal any change related to the treatment with the test item.
- Males
Ulcerations in the forestomach were found in one male at the dose level of 60 and two males at the dose level of 90 mg/kg bw/day. Erosion in forestomach was found in two males at the dose level of 90 mg/kg bw/day. Acanthosis and/or parakeratosis and/or chronic inflammation was seen in two males at the dos level of 30 mg/kg bw/day, two males at the dose level of 60 mg/kg bw/day and six males at the dose level of 90 mg/kg bw/day. Findings in 2 males corresponded to a focus on mucosa and crateriform retraction found in these animals during necropsy.
Necrosis was observed in the trachea of one male at the dose level of 30 mg/kg bw/day four males at the dose level of 60 mg/kg bw/day and four males at the dose level of 90 mg/kg bw/day.
Alveolar edema was found in one male at the dose level of 30 mg/kg bw/day and one male at the dose level of 60 mg/kg bw/day. Alveolar emphysema was found in three males at the dose level of 60 mg/kg bw/day and one male at the dose level of 90 mg/kg bw/day. This finding was correlated with incompletely collapsed or noncollapsed lungs observed during the necropsy.
- Females
Ulcerations in the forestomach were found in one female at the dose level of 90 mg/kg bw/day. Acanthosis and/or parakeratosis and/or chronic inflammation was seen in three females at the dose level of 90 mg/kg bw/day.
Necrosis was observed in the trachea of one female at the dose level of 60 mg/kg bw/day. Several findings during the histological examination in males or females were considered to arise post/peri-mortem (e.g. congestion observed in several organs of animals found dead), to be a result of stress conditions (e. g. lymphoid depletion in some animals or diffuse vacuolation of the adrenal cortex in one male treated at the dose level of 90 mg/kg bw/day) or to be a hemodynamic change (e.g. minimal hemopoiesis or increased hemopoiesis seen in the liver and spleen of females treated with 90 and 60 mg/kg bw/day). These findings were considered to be of no toxicological relevance.

HEMATOLOGY (PARENTAL ANIMALS)
At the dose level of 90 mg/kg bw/d, a statistically significant increase of platelets count was observed in males, whereas only a slight increase was noticed in females. Both these observations were considered to be possibly related to the irritative and degenerative changes in intern organs (stomach and respiratory tract) of the animals.

BIOCHEMISTRY (PARENTAL ANIMALS)
- Males
Test item-related changes of biochemical parameters of the blood serum were observed in males at the dose levels of 90 and 60 mg/kg bw/day.
At the dose levels of 90 and 60 mg/kg bw/day a statistically significant decrease of the urea level was noted. Mean values were 5.71 and 5.64 mmol/l at the dose levels of 90 and 60 mg/kg bw/day, respectively, compared to 7.85 mmol/l in the control group. Level of alkaline phosphatase was also decreased at mid and high dose levels. Mean levels of alkaline phosphatase were 86.4 and 92.6 U/l at the dose levels of 90 and 60 mg/kg bw/day, respectively, compared to 124.3 U/l in the control group. At the dose level of 90 mg/kg bw/day a concentration of total protein in blood serum was reduced as a result of a lower concentration of albumin. Mean concentration of protein (albumin) was 61.71 g/l (40.45 g/l) at the high dose level compared to 65.93 g/l (44.50 g/l) in the control group. These changes were considered to possibly be secondary to the pathological changes in the internal organs, e.g. to the irritated digestive tract, and therefore were considered to be test item-related.
- Females
Test item-related changes of protein concentration were observed in females at the dose level of 90 mg/kg bw/day.
A statistically significant reduction of total protein concentration in blood serum at the high dose level was accompanied by a lower concentration of albumin (not statistically significant). Mean concentration of protein (albumin) was 60.01 g/l (37.75 g/l) at the high dose level compared to 64.49 g/l (40.75 g/l) in the control group. Similar to the effects observed in males, this change was considered to possibly result from the pathological changes in the internal organs, e.g. irritated digestive tract, and therefore were considered to be test item-related.

NEUROBEHAVIORAL EXAMINATIONS (PARENTAL ANIMALS)
During the functional observational battery test item-related effects were noted in males at the dose levels of 30, 60 and 90 mg/kg bw/day and in females at the dose level of 90 mg/kg bw/day. In males, number of rearings was decreased in a dose-dependent manner. The decrease of the number of rearings was observed in 2, 3, 4 and 5 males at the dose levels of 0, 30, 60 and 90 mg/kg bw/day, respectively. At the dose levels of 60 and 90 mg/kg bw/day reduced activity was observed in 3 and 1 male, respectively. At the dose level of 90 mg/kg bw/day 3 males had rales and one of them had also ruffled fur and loose faeces. In two females at the dose level of 90 mg/kg bw/day decreased number of rearings was observed. One of these females had also rales and ruffled fur.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
fertility and reproduction
Effect level:
90 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on a prolongation of mean precoital time that was related to the poor general physical condition of the animals induced by the irritative properties of the test material.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING)
Viability index (number of pups alive on day 4 post-partum as a percentage of pups born alive) was 99.2%, 99.0%, 100.0% and 96.5% in order of ascending dose levels.
At the dose level of 90 mg/kg bw/day a slightly higher postnatal loss was observed. Total number of pups lost was 3 (in 3 litters) compared to 1 pup lost in the control group. Viability index remained in the range of the historical control data (containing values from 91.3% to 100.0%) and therefore higher incidence of post-natal loss at the dose level of 90 mg/kg bw/day was considered not to be test item-related but a result of biological variability.

EXTERNAL EXAMINATION (OFFSPRING)
No abnormal findings were noted at first litter check or during the first 4 days post-partum in any dose group.

SEX RATIOS (OFFSPRING)
Pups sex ratios at first litter check were not affected by exposure to the test item at any dose level.
At first litter check percentages of male pups were 51%, 50%, 46% and 41%, in order of ascending dose level.

BODY WEIGHT (OFFSPRING)
Pups body weights and body weight gain during the first four days post-partum were not affected by exposure to test item at any dose level.
At the dose levels of 0, 30, 60 and 90 mg/kg/day, mean body weights of pups on day 1 postpartum were: 5.8 g, 5.7 g, 6.1 g and 6.0 g, respectively whereas body weight gain of pups during lactation (until day 4 post-partum) were: +49.1%, +50.0%, +46.7% and +49.2%, respectively.

EXTERNAL AND VISCERAL EXAMINATION OFFSPRING)
During external and visceral examination of pups, findings were noted in:
8% examined pups (in 40% litters) in the control group
4% examined pups (in 25% litters) at the dose level of 30 mg/kg bw/day
2% examined pups (in 11% litters) at the dose level of 60 mg/kg bw/day
7% examined pups (in 38% litters) at the dose level of 90 mg/kg bw/day
Type and frequency of the findings did not indicate any test item-related effects at any dose level.

SKELETAL EXAMINATION - Abnormalities and variations (OFFSPRING) See attachment: C48863_Macroscopical examination of pups
During skeletal examination of pups, findings were noted in:
16% examined fetuses (in 50% litters) in the control group
19% examined fetuses (in 38% litters) at the dose level of 30 mg/kg bw/day
4% examined fetuses (in 22% litters) at the dose level of 60 mg/kg bw/day
8% examined fetuses (in 25% litters) at the dose level of 90 mg/kg bw/day
Type and frequency of observed variations did not indicate any test item-related effects at any dose level.
No dose-related abnormalities were found.

SKELETAL EXAMINATION - Stage of Development and Common Variants (OFFSPRING) See attachment: C48863_Macroscopical examination of pups
At the dose level of 90 mg/kg bw/day a statistically significantly (when calculated on both, pups and litter basis) higher number of incompletely ossified os frontale was observed. Percentages of pups (litters) affected with incompletely ossified left/right os frontale were 46%(100%)/54%(88%) at this dose level compared to 19%(44%)/11%(44%) in the control group. This effect was considered to be test item-related. Under the conditions of this study no other signs of a delay in pup development were observed. Therefore a specific effect of the test item on development of fetal/pups skeleton and cranial bones in particular could not be excluded. However, decrease in ossification stage is considered as transient and recovering during the early post natal life, not likely to affect further development and life of the animal. Therefore this effect was considered not to be adverse.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
Development
Generation:
F1
Effect level:
90 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Decrease in ossification stage is considered as transient and recovering during the early post natal life, not likely to affect further development and life of the animal. Therefore this effect was considered not to be adverse.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 7.8.1/4: Summary of performance

Group

(mg/kg bw/d)

1

(0)

2

(30)

3

(60)

4

(90)

Number of females paired

10

10

10

10

Number of females mated

10

10

10

10

Number of females not pregnant

0

1

0

2

Number of females, which did not deliver any pups

0

0

1

0

Number of females, which lost their litters

0

1

0

0

Number of females which reared their pups until day 4 post-partum

10

8

9

8

 

Table 7.8.1/5: Number of females mated during the first pairing period

Day of the pairing period

Group 1

0 mg/kg bw/d

Group 2

30 mg/kg bw/d

Group 3

60 mg/kg bw/d

Group 4

90 mg/kg bw/d

1

1

2

3

1

2

5

4

3

1

3

1

2

4

3

4

2

2

-

2

8

-

-

-

1

12

-

-

-

1

13

1

-

-

1

Median precoital time

2

2

2

4

Mean precoital time

3.5

2.4

2.1

5.3

N

10

10

10

10

Table 7.8.1/6: Incidence and Mean severity of Main findings in stomach, liver and spleen

 

 

Males

Females

Organ

Group

1

2

3

4

1

2

3

4

Stomach

Acanthosis/Mean Grade

-

2/2.0

2/2.5

6/3.3

-

-

-

2/2.5

Parakeratosis/Mean Grade

-

1/2.0

2/2.0

6/2.5

-

-

-

2/2.0

Inflammation/Mean Grade

-

2/1.5

2/3.0

6/3.3

-

-

-

3/2.0

ForestoEros./Mean Grade

-

-

-

2/1.0

-

-

-

-

ForestoUlcer./Mean Grade

-

-

1/2.0

2/2.0

-

-

-

1/2.0

Liver

Liver Hemopoiesis/Mean grade

-

-

-

-

-

-

2/1.0

2/1.0

Spleen

Spleen Incr. Hemopoiesis/Mean Grade

-

-

-

-

2/1.5

1/1.0

4/2.3

4/1.5

 

Applicant's summary and conclusion

Conclusions:
Based on a prolongation of mean precoital time that was related to the poor general physical condition of the animals induced by the irritative properties of the test material, the dose level of 90 mg/kg bw/day was considered to be the NOAEL for reproduction and breeding.
Based on a delay in the ossification state of cranial bones observed in pups at the dose level of 90 mg/kg bw/day, the NOAEL for pup development was determined to be 90 mg/kg bw/day even if this effect is considered to be of low level of concern.
Executive summary:

A Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test was conducted according to the OECD Guideline No. 422 and in compliance with GLP. The substance was dosed once daily by gavage at dose levels of 0, 30, 60 and 90 mg/kg bw/day (dose volume of 4 mL/kg bw in corn oil) to Han Wistar rats. Male rats were administered for at least 28 days and female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post-partum.

During the treatment ten animals were found dead before scheduled necropsy: four males at the dose level of 90 mg/kg bw/day, four males and one female at the dose level of 60 mg/kg bw/day and one male at the dose level of 30 mg/kg bw/day. Some of these animals had reduced food consumption and reduced body weight gain. Because of breathing noises observed in these animals a penetration of the test item into the airways was assumed. During histopathological examination necrosis of the trachea was found in all these animals and this pathological change was identified as a cause for the deaths.

Treatment with the test item caused bedding in mouth, salivation and breathing noises in several males and females at the dose levels of 90 and 60 mg/kg bw/day, at the dose level of 30 mg/kg bw/day breathing noises were observed in one male. These clinical signs indicated irritative properties of the test item.

During functional observational battery test item-related effects on number of rearings and locomotor activity were recorded in males at the dose levels of 90, 60 and 30 mg/kg bw/day and in females at the dose level of 90 mg/kg bw/day.

Food consumption, body weights and body weight gain were adversely reduced in males treated at the dose level of 90 and 60 mg/kg bw/day. In females reduction of food consumption was observed at the dose level of 90 mg/kg bw/day. Because no test item-related effects on body weights or body weight gain were observed, reduction of food consumption in females was considered not to be adverse.

As established during macroscopical and microscopical examinations treatment with the test item induced lesions in the forestomach of males treated at the dose levels of 30, 60 and 90 mg/kg bw/day and in females treated at the dose level of 90 mg/kg bw/day. These inflammatory, degenerative and reactive changes were caused by irritative or corrosive properties of the test item. Residual microdroplets of the dose formulations remaining on intubation cannula during removal after administration could irritate esophagus, cause an opening of the epiglottis and aspiration of the test item. This was assumed to be the reason for the macroscopical and microscopical changes found in the respiratory tract of the animals.

 

With the exception for mean precoital time, the relevant reproduction parameters (duration of gestation, number of corpora lutea, implantation sites, pre- and post implantation losses, number of pups) were not affected by the treatment with the test item. The litter size at first litter check and on day 4 post-partum was also not affected.

A prolongation of mean precoital time was recorded at the dose level of 90 mg/kg bw/day. In the absence of any test item-related pathological changes in reproductive organs in males and in females, prolongation of the precoital time was considered to be secondary to the bad condition of males and females during the pairing period and not to be a sign of test item-related toxicity to reproduction. Because this effect did not result in lower number of offspring, this effect was considered not to be adverse.

No effects on body weights and body weight gain of pups during the first four days post-partum were observed.

External and visceral examination of pups did not give any indication of test item-related effects on development.

During skeletal examination, an increase of incompletely ossified os frontale was observed at the dose level of 90 mg/kg bw/day. The authors considered that this effect was test item-related. However, the delayed of ossification observed in the fetuses from the dams treated at the highest dose were likely secondary to the maternal toxicity. Furthermore, in the absence of any further signs of a delay in pup development and because a delay in the ossification is expected to recover during the post natal life and not impact the later development and animal life, this effect was not considered as adverse.

In conclusion, findings in parental animals observed during the in live-phase and recorded upon termination of the study were considered to result from irritative or corrosive properties of the test item. Under the conditions of this study no indications of systemic toxicity of the test item were observed.

Consequently, because of one case of death at the low dose level due to the irritative/corrosive effects resulting from gavage administration, no NOAEL (No Observed Adverse Effect Level) could be established for general toxicity for parental animals.

Based on a prolongation of mean precoital time that was related to the poor general physical condition of the animals induced by the irritative properties of the test material, the dose level of 90 mg/kg bw/day was considered to be the NOAEL for reproduction and breeding.

Based on a delay in the ossification state of cranial bones observed in pups at the dose level of 90 mg/kg bw/day, the NOAEL for pup development was considered to be 90 mg/kg bw/day. However, the slight ossification retardation observed at the highest dose levels in this study is considered to be of low level of concern as nonlethal and not detrimental to postnatal survival variations which are generally reversible or transitory. In fact, the delays in ossification resolve in time as the growth of the individual reaches a comparable stage to that of the control group to which it is being compared.