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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 09 FEB 2012 and 03 JUL 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 422) and in compliance with GLP.
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxocity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

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, Inc., Maasheseweg 87c, 5800 AN Vernay / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 315 to 369 g (males) and 187 to 243 g (females)
- Identification: parent animals had cage card and individual animal number (ear tattoo), pups were individually tattooed with Indian ink: on day 1 post partum.
- 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.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was a 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands) batch/lot nos. 02105111001, 02105111201, 100099 and S211008A63972). 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 (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Results of representative analyses for contaminants were included in the report as an Appendix.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples were included in the report as an Appendix.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1.0% CMC / 0.05% Tween 80 in highly purified water
Details on exposure:
DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

Test item was weighed into a glass beaker on a tared precision balance. Appropriate amount of the vehicle was weighted and added to the test item (w/w). Dose formulations were mixed on a magnetic stirrer for approximately 2 hours until mixtures were homogeneous. Separate formulations were prepared for each concentration.

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

STORAGE OF DOSE FORMULATIONS

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

Based upon the results of stability analyses performed within the non GLP Harlan Laboratories study D44836 14-Day Oral Toxicity (Gavage) Study in the Wistar Rat, dose formulations are stable for at least 7 days if stored at room temperature.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg bw/day (control group), 100 mg/kg bw/day (group 2), 300 mg/kg bw/day (group 3) and 1000 mg/kg bw/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D44836, using dose levels of 0, 100, 300 and 1000 mg/kg/day, where no adverse effects were observed up to the highest dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL (control group), 10 mg/mL (group 2), 30 mg/mL (group 3) and 100 mg/mL (group 4)
- Duration of Acclimatization Period: 7 days
- Duration of Treatment Period: 32 days males and approximately 7 weeks females
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 of 2 g each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (7 days) samples of about 2 g of each concentration were taken from the middle only of each aliquot used on day 7 of the treatment. During the sixth week of the treatment samples of about 0.5 g were taken to repeat the measurement of concentration, homogeneity and stability (7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and stored at -20 ± 5 °C until analysis.

The samples were analyzed by UV-VIS measurement following an analytical procedure developed by Harlan Laboratories. The test item was used as the analytical standard.

Following results were obtained:

Blank samples showed no absorbance and, therefore, it was confirmed that only 1.0% CMC / 0.05% Tween 80 in highly purified water applied within the control experiment.

The application formulations investigated during the study were found to comprise test item in the range of 83.0% to 111.3% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of the test item in the preparations was approved because single results found did not deviate more than 9.8% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept seven days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean, except for group 2 and 3 prepared on 16-Feb-2012 that exceeded the acceptance criteria. In application formulations of group 2 (10 mg/mL), the maximum deviation of time-zero mean was found to be 11.8%, and in application formulations of group 3 (30 mg/mL), the maximum deviation of time-zero mean was found to be 35.8%. However, there are some hints of what could be the cause. On the first and 7th treatment day as well as backup samples (date of preparation 16-Feb-2012), erroneously high amount of samples (about 2 g instead 0.5 g) were taken for analytical measurements making it difficult to work up. That was considered to probably be the reason for the differences as a second time using small amount of sample (0.5 g) shows the homogeneity and stability of the application formulation.

In conclusion, the results indicate the accurate use of the test item and 1.0% CMC / 0.05% Tween 80 in highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
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 positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

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

32 days

FEMALES

about 7 weeks
Frequency of treatment:
once daily
Duration of test:
MALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment End: On day before sacrifice
- Blood Sampling: At Termination
- Necropsy: After 32 days of treatment, when no longer needed for assessment of reproductive effects

FEMALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment End: On day 4 post partum
- Blood Sampling: On day 5 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle

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

Females: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period; on days 0 - 7, 7 14 and 14 - 21 during gestation period and on days 1 - 4 of during lactation period.

No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

DETAILED CLINICAL OBSERVATIONS

Detailed clinical observations were performed outside the home cage in all animals once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.

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 behavior were also reported.

FUNCTIONAL OBSERVATIONAL BATTERY

At one time during the study, on day 4 post partum, relevant parameters were performed with 5 P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
- Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
- Hand-held observations: muscle tone, constituation, skin, pupile 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, behavior, 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.

Any abnormal findings were recorded and, where appropriate, graded in severity.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS

Blood samples were obtained from 5 lactating females of each group at the end of the pre-pairing period. Blood samples were drawn from the retro-orbital plexus of all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The following hematology parameters were determined:
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count
- Platelet count
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:
- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

TERMINATION AND NECROPSY

Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

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

ORGAN WEIGHTS

At the scheduled sacrifice, from 5 females sacrificed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Thymus
- Spleen

TISSUE PRESERVATION

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries

In addition, from 5 females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Gross lesions
- Brain (representative regions including cerebrum, cerebellum and pons)
- Spinal chord
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Uterus (with vagina)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Bone marrow

HISTOTECHNIQUE

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

HISTOPATHOLOGY

Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions.

Histological examination of ovaries was carried out on any females that did not give birth.

A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator for inclusion in the main report as an appendix.
Ovaries and uterine content:
The ovaries and uterus were examined after termination on day 5 post partum. Examinations included number of corpora lutea and number of implantation sites.
Fetal examinations:
Not performed.
Statistics:
The following statistical methods were used to analyze food consumption, body weights, reproduction data, grip strength, landing foot splay, body temperature, locomotor activity, hematology and biochemistry parameters, urinalysis, sperm analyses and organ weights:
- Means and standard deviations of various data were calculated and included in the report.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can 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 cannot be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables can be dichotomized without loss of information.
Indices:
From the on-line recorded reproduction data, the following parameters were calculated: mean precoital time, percentage mating, fertility index, conception rate, post-implantation loss, gestation index, birth index, dead/live pups at first litter check, pups sex ratio and viability index.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
1. IN-LIVE DATA

VIABILITY / MORTALITY

All animals survived scheduled study period.

DAILY CLINICAL SIGNS OR OBSERVATIONS

Feces stained yellow were noted in all females in all dose groups starting from day 2 or 3 of the treatment until completion of the study. This observation was due to staining properties of the test item.

No further test item-related clinical signs or observations were noted females at any dose level.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS

No test item-related findings were noted during weekly detailed clinical observations.

Incidentally, in one female at the dose level of 1000 mg/kg bw/day missing upper incisors on day 6 and 13 of the gestation period were recorded.

No further findings were noted in males or females in any dose group.

FUNCTIONAL OBSERVATIONAL BATTERY

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.

All findings recorded during the tests were considered not to be test item-related. Decreased or increased rearings, increased faeces balls and vocalization were noted in individual animals without dose dependency. At the dose level of 1000 mg/kg bw/day, one female was noted with multiple findings: reduced activity, ruffled fur, decreased rearings and salivation. Because of isolated occurrence, this observation was considered not to be related to the treatment.

Remaining parameters under investigation were similar in all groups.

LOCOMOTOR ACTIVITY

No effects on locomotor activity were noted in females at any dose level.

Mean beam counts during the 30 minutes of measurement at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 965, 917, 853 and 867 in females.

FOOD CONSUMPTION

No effects on food consumption were noted in females at any dose level.

Incidentally, statistically significantly higher food consumption was noted at the dose level of 100 mg/kg bw/day on days 8 - 11 of the pre-pairing period and at the dose level of 300 mg/kg bw/day on days 7 - 14 of the gestation period. In the absence of any dose dependency, these differences were not related to the treatment.

Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +8.0%, ±0.0%, and +3.4% during the pre-pairing period, +1.8%, +6.3% and +3.1% during the gestation period and +11.7%, -3.0% and +3.8% during the lactation period (percentages refer to the respective values in the control group).

BODY WEIGHTS

Body weights and body weight gain of females were not affected by the treatment with the test item at any dose level.

Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 8%, 9%, 8% and 8% during the pre-pairing period, 53%, 53%, 59% and 57% during the gestation period and 6%, 7%, 6% and 6% during the lactation period (percentages refer to the body weight change within the respective period).

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

No effects on hematology parameters were noted in females at any dose level.

CLINICAL BIOCHEMISTRY

No test item-related effects on biochemistry parameters were noted in females at any dose level.

Following parameters were statistically significantly lower if compared to the respective control values: concentration of albumin at the dose level of 100 mg/kg bw/day, concentration of albumin and albumin to globulin ratio at the dose level of 300 mg/kg bw/day and concentration of total protein and albumin at the dose level of 1000 mg/kg bw/day. Changes of these values were not dose depended and all values were within historical control range. Therefore these changes were considered not to be test item-related.

3. TERMINAL FINDINGS

ORGAN WEIGHTS

No significant differences in absolute or relative organ weights were noted in females at any dose level.

MACROSCOPICAL FINDINGS

Type and distribution of findings noted during macroscopical examination of females did not indicate any test item related effect.

HISTOPATHOLOGY FINDINGS

Under the conditions of this experiment, test item did not induce histopathological lesions. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

During examination of reproduction organs of infertile females no findings correlated to the infertility were noted in females.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
For pup data see chapter: Any other information on results incl. tables

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

1. REPRODUCTION, BREEDING AND PUP DATA  

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litters

 

Group
(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(1000)

Female numbers

45-55

56-66

67-77

78-88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of non pregnant females (A)

2

1

1

2

Numbers of pregnant females,
which did not deliver any pups (B)

0

0

0

1

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

9

10

10

8

(A) Female nos. 46, 50, 59, 69, 83 and 85. (B) Female no. 87.

 

MATING PERFORMANCE AND FERTILITY

  

Mating performance and fertility were not affected by the treatment at any dose level.

  

Percentage of mating was 100% in all groups. Mating of all females was recorded during the first seven days of the pairing period.

 

Mean (median) precoital times were 2.9 (3), 2.6 (3), 2.9 (3) and 2.8 (3) days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

Six females were not pregnant: two in the control group, one at each dose level 100 and 300 mg/kg bw/day and two at the dose level of 1000 mg/kg bw/day. Consequently, fertility indexes (number of females pregnant as percentages of females paired) and conception rate (number of females pregnant as percentages of females mated) were 81.8%, 90.9%, 90.9% and 81.8% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

One female at the high dose level had two implantation sites but delivered no pups. Consequently, gestation index (number of females with living pups as percentages of females pregnant) was 88.9% at the high-dose level and 100% in remaining groups.

 

CORPORA LUTEA COUNT

  

No test item-related effects on corpora lutea count were observed at any dose level.

 

Mean number of corpora lutea per dam was 13.6, 15.5, 17.4 and 14.4 in order of ascending dose levels.

 

At the dose level of 300 mg/kg bw/day, mean number of corpora lutea per dam was statistically significantly higher when compared to the control group. In the absence of any dose dependency, this observation was considered not to be related to the treatment.

 

DURATION OF GESTATION

  

No effects on duration of gestation were observed at any dose level.

 

Mean duration of gestation was 21.6, 21.8, 21.8 and 21.6 days, in order of ascending dose level.

 

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

 

No effects on implantation rate and post-implantation loss were observed at any dose level.

 

In order of ascending dose levels, mean number of implantations was 13.0, 13.0, 14.2 and 14.4 per dam whereas mean incidence of post-implantation loss was 1.0, 1.0, 1.4 and 0.4 per dam.

 

LITTER SIZE AT FIRST LITTER CHECK

 

No effects on litter size were noted at any dose level.

During the first litter check, one dead pup was found in a litter at the dose level of 1000 mg/kg bw/day. Because of isolated occurrence, this finding was considered to be incidental.

 

Mean number of living pups per dam at first litter check was 12.0, 12.2, 12.8 and 14.1 in order of ascending dose levels.

Birth index (number of pups born alive as a percentage of implantations) was 92.3%, 92.2%, 90.1% and 97.0% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.

 

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

 

No test item-related effects on postnatal loss were noted at any dose level.

 

In the control group one pup was missing on day 4, at the low-dose level one pup was missing on day 2, at the mid dose level eight pups (from 2 litters) were missing on day 4 and at the high dose level two pups (from two litters) were missing on day 2 of the lactation period. Mean postnatal loss during four days of lactation was 0.1, 0.1, 0.8 and 0.3% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Consequently, viability index (number of pups alive at termination on day 4 p.p. as a percentage of pups born alive) was 99.1, 99.2, 93.8 and 98.2 in order of ascending dose levels.

 

At the dose level of 300 mg/kg bw/day, total number of pups lost was statistically significantly higher and viability index was statistically significantly lower when compared to the respective control values. These observations were due to a loss of seven pups in one litter. Because higher mortality of pups was noted only in one litter and in the absence of increased mortality of pups at the dose level of 1000 mg/kg bw/day, this observation was considered not to be related to the treatment.

 

EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

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

 

In the control group, missing tail tip was noted at first litter check and during lactation in one pup. At the dose level of 300 mg/kg bw/day, all pups in one litter had reduced temperature on day 3 and several pups from this litter had reduced temperature on day 4 of the lactation period. At the dose level of 1000 mg/kg bw/day, one pup, which was dead at first litter check had no milk in the stomach and was partially cannibalized.

 

No further findings were noted in pups at any dose level.

 

SEX RATIOS OF PUPS

 

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 48%, 48%, 38% and 49% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.

 

BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUM

 

Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

 

Mean body weights of pups on day 1 post partum were: 8.9 g, 9.2 g, 8.9 and 8.2 g and mean differences in body weights during lactation were +47.4%, +47.7%, 43.6% and +38.7%, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively.

 

At the dose level of 1000 mg/kg bw/day, slightly not statistically significantly lower body weight gain of pups was noted. This effect was considered to be due to a higher number of pups at this dose level which was supported by observation that reduction of body weight gain was more pronounced in litters of higher size. Therefore this effect was not test item-related.

 

MACROSCOPICAL FINDINGS IN PUPS

 

No findings were found during necropsy of pups in any dose group.

 

2. IN-LIVE DATA OF PARENTAL MALES

 

VIABILITY / MORTALITY

 

All animals survived scheduled study period.

 

DAILY CLINICAL SIGNS OR OBSERVATIONS

 

Feces stained yellow were noted in all males in all dose groups starting from day 2 or 3 of the treatment until completion of the study. This observation was due to staining properties of the test item.

 

No further test item-related clinical signs or observations were noted in males or females at any dose level.

 

Incidentally, scabs on the neck were noted in one male in the control group.

 

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS

 

No findings were noted during weekly detailed clinical observations in males.

 

FUNCTIONAL OBSERVATIONALBATTERY

 

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.

 

All findings recorded during the tests were considered not to be test item-related. Decreased or increased rearings, increased faeces balls and vocalization were noted in individual animals without dose dependency. Remaining parameters under investigation were similar in all groups.

 

LOCOMOTOR ACTIVITY

 

No effects on locomotor activity were noted in males at any dose level.

 

Mean beam counts during the 30 minutes of measurement at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 1079, 1198, 1252 and 1207.

 

FOOD CONSUMPTION

 

No effects on food consumption were noted in males at any dose level.

 

Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: ±0.0%, -1.2% and -4.6% during the pre-pairing period and -2.0%, -2.0% and -0.8% during the after pairing period (percentages refer to the respective values in the control group).

 

BODY WEIGHTS

 

At the dose levels of 1000 and 300 mg/kg bw/day, reduction in body weight gain was noted during the pre-pairing period; mean body weight gain within this period was +10% and +12% at the high- and mid-dose level respectively, compared to +14% in the control group. Reduction in body weight gain was statistically significant at the high-dose level starting from day 3 until the end of pre-pairing period and at the mid-dose level on days 7, 9, 11, 12 and 14 of the pre-pairing period. This effect was considered to be test item-related. During the pairing and after pairing periods, body weight gain was similar at all dose levels.

Body weights of males in all dose groups were similar to the respective control values during the entire study period.

Because the reduction in body weight gain at the high- and mid-dose levels was reversible and did not result in any significant differences in body weights, this observation was considered not to be adverse.

 

Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: +14%, +13%, +12% and +10% during the pre-pairing period, +3%, +2%, +3% and 3% during the pairing period and +1%, +1%, +1% and +2% during the after pairing period (percentages refer to the body weight change within the respective period).

 

2. CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL MALES

 

HEMATOLOGY

 

No test item-related effects on hematology parameters were noted in males at any dose level.

 

At the dose level of 300 mg/kg bw/day, amount of large unstained cells (LUC) was statistically significantly lower when compared to the control value. No reduction of this value occurred at the dose level of 1000 mg/kg bw/day and therefore the difference at mid-dose level was not related to the treatment.

 

CLINICAL BIOCHEMISTRY

 

No test item-related effects on biochemistry parameters were noted in males at any dose level.

 

At the dose level of 300 mg/kg bw/day, potassium concentration was statistically significantly higher when compared to the control value. No increase of this value occurred at the dose level of 1000 mg/kg bw/day and therefore the difference at mid-dose level was not related to the treatment.

 

URINALYSIS

 

No changes in urine parameters were noted in males at any dose level.

 

3. TERMINAL FINDINGS OF PARENTAL MALES

 

SEMINOLOGY AND SPERMATID COUNT

 

At the dose levels of 1000 and 300 mg/kg bw/day, statistically significant changes in motility of sperms were noted. Mean count of progressive sperms was reduced at the high dose level and it was 60.4% compared to 75.6% in the control group. This value is within the range of a limited pool of historical controls for OECD 422 (n=4) extended by OECD 416 data (n=5). Mean count of not motile sperms was increased and was 35.5% and 34.2% at the high- and mid-dose dose levels, respectively, compared to 20.2% in the control group. These changes might be test item-related. However no significant dose dependent trend indicated by probability values of <0.05 was determined for any of these changes when performing a linear regression analysis (least squares). In the absence of any findings during necropsy or histopathological examination of male reproductive organs as well as absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

 

No further changes were noted during sperm analysis. At the high-dose level, all morphological categories of sperms were represented with similar frequency to that in the control group whereas sperm count was similar (testis) or slightly higher (epididymidis) when compared to the respective control values.

 

ORGAN WEIGHTS

 

No test item-related changes in organ weights were noted in males at any dose level.

 

At the dose level of 100 mg/kg bw/day, statistically significantly lower weight of testis was noted. This effect was due to reduced testis weights of one male (no. 15, malformation of testis in this male correlated with histopathological findings). No significant changes of testis weights were noted at the mid- and high dose levels and therefore these changes were not related to the treatment with the test item.

 

No further significant differences in absolute or relative organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS

 

Type and distribution of findings noted during macroscopical examination of males did not indicate any test item related effect.

 

HISTOPATHOLOGY FINDINGS

 

Under the conditions of this experiment, the test item did not induce histopathological lesions. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

There were no abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of cell populations. Individual lesions recorded were within the range of background alterations that may be recorded in this type of study, in rats of this strain and age.

 

During examination of reproduction organs of infertile males, bilateral total tubular degeneration of testes was found in male no. 15 at the dose level of 100 mg/kg bw/day. This lesion was considered to be the reason for infertility of male no.15. No further findings correlated to the infertility were noted in reproduction organs of the remaining infertile males or females.

 

Applicant's summary and conclusion

Conclusions:
This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats. The test item was administered in vehicle (1.0% CMC / 0.05% Tween 80 in highly purified water) at dosages of 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 32 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Under the conditions of this study, no adverse effects were found in males or females up to the highest dose level of 1000 mg/kg bw/day.

All animals survived the scheduled study period.
During the treatment, faeces stained yellow were noted in all males and females receiving test item. This observation was due to staining properties of the test item.

At the dose levels of 1000 and 300 mg/kg bw/day in males, statistically significantly reduced body weight gain was noted during the pre-pairing period. During the remaining study period body weight gain at the high- and mid-dose levels was similar to the control values. Body weights at these dose levels were similar to the control values during the entire study period. Because reduction of body weight gain was reversible and no statistically significant differences in body weights were noted at the high- and mid-dose levels, this effect was considered not to be adverse.

No further test item related observations were noted in males or females at any dose level during the live part of the study.

Terminal examinations revealed changes in motility of sperms at the dose levels of 1000 and 300 mg/kg bw/day. Statistically significant reduction in mean count of progressive sperms and increase in mean count of not motile sperms were noted at the high dose level and statistically significant increase in mean count of not motile sperms was noted at the mid-dose level. No further effects on male reproductive system were noted during the study. Sperm morphology and sperm count at the high-dose level was similar to the control values. Weights of male reproductive organs, macroscopical and histopathological examination of testes and epididymides gave no indication of any treatment-related effect. Further, no indication of effects on reproduction was noted within this study up to and including the highest dose level. For this reason, changes in motility of sperms were considered not to be adverse.

Reproduction and development were not affected by the treatment. Mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss or litter size were similar in the control and all dose groups. There were no test item-related findings in pups noted during the first litter check, the first 4 days post partum or during the necropsy, pups body weights and body weight gain were not affected by the treatment at any dose level.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level used.
Executive summary:

The purpose of this study was to generatepreliminaryinformation concerning the effects of tets item on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

The test item was administered to male rats for 32 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels were applied:

 

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 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (1.0% CMC / 0.05% Tween 80 in highly purified water).

 

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 

All animals survived the scheduled study period.

 

During the treatment, yellow stained faeces was noted in all males and females in dose groups. This observation was due to staining properties of the test item.

 

No further test item-related observations were noted during clinical daily or detailed weekly observations in males or females at any dose level.

 

FUNCTIONAL OBSERVATIONAL BATTERY AND LOCOMOTOR ACTIVITY OF PARENTAL ANIMALS

 

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.

 

Locomotor activity was not affected by the treatment with test item in males or females at any dose level.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 

No effects on food consumption were noted in males or females at any dose level.

 

BODY WEIGHT OF PARENTAL ANIMALS

 

In males at the dose levels of 1000 and 300 mg/kg bw/day, dose dependent and statistically significant reduction in body weight gain was noted during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because reduction in body weight gain at the high- and mid-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.

 

Body weights and body weight gain of females were not affected by the treatment at any dose level.

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

 

No test item-related effects on hematology and clinical biochemistry parameters were noted in males or females at any dose level.

 

No changes in urine parameters were noted in males at any dose level.

 

REPRODUCTION AND BREEDING DATA

 

Mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss, litter size or postnatal loss were not affected by the treatment with the test item.

 

SEMINOLOGY AND SPERMATID COUNT IN PARENTAL ANIMALS

 

At the dose level of 1000 mg/kg bw/day, reduction in mean count of progressive sperms and increase in mean count of not motile sperms were noted. At the dose level of 300 mg/kg bw/day, increase in mean count of not motile sperms was noted. A significant dose dependent trend couldn't be established.

In the absence of any findings during necropsy or histopathological examination of male reproductive organs as well as absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

Sperm morphology and sperm count were not affected by the treatment at any dose level.

 

ORGAN WEIGHTS OF PARENTAL ANIMALS

 

No test item-related changes in organ weights were noted in males or females at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

 

Type and distribution of findings noted during macroscopical examination did not indicate any test item related effect.

All findings recorded during histopathological examination were within the range of normal background lesions which may be recorded in animals of this strain and age.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

No test item-related findings were noted in pups during first litter check and 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

 

Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

 

MACROSCOPICAL FINDINGS IN PUPS

 

No findings were found during necropsy of pups in any dose group.

 

CONCLUSION

 

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