Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
Remarks:
No deviations ocurred that negatively impacted the integrity of the study.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M Company, Lot 3
- Expiration date of the lot/batch: 17 June, 2004
- Purity test date:
30 January, 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature in the dark.
- Stability under storage conditions:
Stable
- Stability under test conditions:
Stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
The test article was tested as a suspension in propylene glycol and was determined to be stable for at least 4 hours at room temperature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test article was suspended in propylene glycol.
- Final preparation of a solid:
Suspended in propylene glycol.

FORM AS APPLIED IN THE TEST: The test article was suspended in propylene glycol.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species and strain commonly used in reproductive and developmental screening studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland
- Females (if applicable) nulliparous and non-pregnant:Yes
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: Male mean: 250-258 grams, Female mean: 183-186 grams.
- Fasting period before study: None
- Housing: 5 animals per sex per cage. During mating, females were caged together with males on a one-to-one basis. Mated females and males were individually housed in labelled polycarbonate cages containing sawdust as bedding.
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet.
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: 5 days.

DETAILS OF FOOD AND WATER QUALITY: Food was analyzed for nutriets and contaminants on a regular basis. Tap water was analyzed quarterly.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1-23.5 C.
- Humidity (%): 42-87 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 July, 2003 To: 05 September, 2003.
Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a rubber catheter attached to a plastic disposable syringe. Maximum dose volume was 5 mL/kg body weight.
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared daily within 4 hours prior to dosing and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the test substance and vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test article solubility and test system compatibility.
- Concentration in vehicle: To appropriately dose 50, 150 and 1000 mg/kg/day at a maximum dose volume of 5 mL/kg body weight.
- Amount of vehicle (if gavage): 5 mL/kg body weight.
- Lot/batch no. (if required): No data
- Purity: No data.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
The males and females were exposed for 2 weeks prior to mating, aduring mating and up to the day prior to necropsy. Exposure period was at least until the minumum total dosing period of 28 days had been completed.
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control (propylene glycol)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a range-finding study in which dose levels of 150 and 1000 mg/kg were administered for 5 day.
- Rationale for animal assignment (if not random): Random
- Fasting period before blood sampling for clinical biochemistry: Overnight
:
Positive control:
None
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly; Days 1 and 4 of lactation

FOOD EFFICIENCY: Weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to necropsy.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight.
- How many animals: 5 per sex
- Parameters checked:

Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Platelet count
Red cell distribution
Total leucocyte count
Differential leucocyte count
Prothrombin time
Partial thromboplastin time

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
immediately prior to necropsy.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight.
- How many animals:
5 per sex
- Parameters checked:

Alanine aminotransferase
Alkaline phophatase
Aspartate aminotransferase
Bilirubin, total
Chloride
Cholesterol, total
Creatinine
Glucose
Phosphorous
Protein, total
Protein, albumin
Triglycerides
Urea
Calcium
Potassium
Sodium

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to necropsy.
- Dose groups that were examined: 5 per sex per group.
- Battery of functions tested: sensory activity, grip strength, motor activity

IMMUNOLOGY: No

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see attached table.

HISTOPATHOLOGY: Yes, see attached table.
Statistics:
If variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.

The Steel-test was applied instead of the Dunnett-test if the data could not be assumed to follow a normal distribution.

The exact Fisher-test was applied for 2x2 tables if variables could be dichotomized without loss of information.

All test were two-sided and in all cases p< 0.05 was accepted as the lowest level of significance.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, the following clinical observations were noted: lethargy, hunched posture, uncoordinated movements, decreased locomotoractivity, ventro-lateral recumbency, quick breathing, labored respiration rates, shallow respiration, swelling of the genital region and abdomen, piloerection, reddiscoloration of urine, diarrhea, salivation, chromodacryorrhea of both eyes and snout, lean appearance, and ptosis. Salivation and diarrhea were alsoobserved at the 150 mg/kg/day dose level.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 1000 mg/kg/day, two females (one found dead, one euthanized in extremis) died due to meningitis and three males (two founddead, one euthanized in extremis) died due to gavage error (1 male) or cause of death not evident (2 males). At 150 mg/kg/day, one female was euthanized inextremis due to a uterine prolapse just after delivery. All deaths are likely to be incidental findings; however, a possible relationship to treatment cannot entirely be excluded.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of 1000 mg/kg/day-treated males were statistically significantly decreasedduring treatment in 70% of animals. Females treated at 1000 mg/kg/day had statistically significantly decreased body weights and body weight gain during thepre-mating period. These females also had decreased body weights during pregnancy and lactation. No abnormal body weight changes among rats treated at50 or 150 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, absolute and relative food consumption were decreased during the complete treatment period. Statistical significancde was reached for food consumption and relative food consumption during pre-mating (males), and for food consumption during post-mating (males) and lactation (females).

No treatment related effect on food consumption and relative food consumption were observed at mid and low dose groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant differences of hematology parameters were recorded in the highest dose group at the end of treatment:

- Decreased neutrophil count (females)
- Increased erythrocyte count (males and females)
- increased hemoglobin concentration (males and females)
- Increased hematocrit (males and females)
- Increased mean corpuscular volume (males)
- Increased mean corpuscular hemoglobin (males)
- Increased mean corpuscular hemoglobin concentration (males)
- Increased red cell distribution width (males)

No other statistically significant effects upon hemotology parameters were noted.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant differences in clinical biochemistry parameters were recorded in the highest dose group at the end of treatment:

- Increased alanine aminotransferase (males)
- Increased alkaline phosphatase (males)
- Increased urea (males)
- Increased chloride (males)
- Increased inorganic phosphorous (males)
- Decreased aspartate aminotransferase (females)
- Decreased bilirubin (females)
- Increased albumin (females)

No other statistically significant effects upon clinical chemistry parameters were noted.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No changes were observed in hearing ability, papillary reflex, static righting reflex and grip strength in the treated animals compared to controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following treatment-related changes were present:

Males: (1000 mg/kg)
- Decreased terminal body weight
- Increased relative liver weight
- Increased relative spleen weight
- Increased relative epididymides weight

Females (1000 mg/kg)
- Decreased terminal body weight
- Increased absolute and relative liver weight
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At necropsy, respectively, three, four, seven and fifteen animals (out of twenty animals) in the control, 50, 150 and 1000 mg/kg dose groups showed abnormalities.

Four males in the highest dose group appeared to have yellowish nodules on the epididiymides. This finding correlated with the microscopic finding of sperm granuloma and was considered to be treatment-related.
Neuropathological findings:
effects observed, treatment-related
Description (incidence and severity):
Meningitis was present in the brain and spinal cord of two females in the 1000 mg/kg/day dose group. These deaths are likely to be incidental findings; however, a possible relationship to treatment could not be entirely excluded. Lethargy, uncoordinated movements, and decreased locomotor activity were observed in the 1000 mg/kg/day dose group. There were no changes in static righting reflex and grip strength in test article treated animals when compared to control group animals.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Primary treatment-related findings were confined to the liver, spleen and epididymides of high dose animals:

Liver: Minimal/slight hepatocyte hypertrophy in males and females
Spleen: Increased severity of hematopoiesis in males and females
Epididymides: Slight/moderate sperm granuloma in males
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No noeplastic lesions were observed upon histopathological examination.
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
neuropathology
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
central nervous system
Organ:
brain
spinal cord
Treatment related:
yes
Dose response relationship:
no
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
other: Epididymides
Treatment related:
yes
Dose response relationship:
no
Conclusions:
Based on the results of the study, the No Observed Adverse Effect Level for the test article is 150 mg/kg/day.
Executive summary:

The subacute and reproductive/developmental toxicity of the test article (off white solid, Lot 3) was evaluated in Wistar rats following daily oral gavage during premating, mating, gestation, and lactation. This study was performed in compliance with OECD GLP(1997).The study method was based on OECD 422 (1996).The test article was prepared in propylene glycol (vehicle) just prior to each dosing. Rats (10/sex/dose)received 0 (vehicle; Group 1), 50 (Group 2), 150 (Group 3), or 1000 (Group 4) mg/kg/day test article via oral gavage at a dose volume of 5 mL/kg.The males and females were exposed for 2 weeks prior to mating and during mating. Females were paired 1:1 with males from the same treatment group. Mating was confirmed by copulation plug absence (designated as Gestation Day 0). Males were treated for at least 28 days total.In addition to exposure during prematingand mating, females were exposed during gestation and for at least 4 days of lactation.The offspring were euthanized on Day 4 of lactation or shortly thereafter.The following parameters were evaluated: clinical observations (at least once daily), functional observations (5/sex/group; prior to termination), body weights(weekly; Days 1 and 4 of lactation), food consumption (weekly), reproductive processes, offspring observations, necropsy (termination), clinical chemistry (termination) and organ weights and/or histopathology of select organs (termination). At 1000 mg/kg/day, two females (one found dead, one euthanized in extremis) died due to meningitis and three males (two found dead, one euthanized in extremis) died due to gavage error (1 male) or cause of death not evident (2 males). At 150 mg/kg/day, one female was euthanized in extremis due to a uterine prolapse just after delivery. All deaths are likely to be incidental findings; however, a possible relationship to treatment cannot entirely be excluded.At 1000 mg/kg/day, the following clinical observations were noted: lethargy, hunched posture, uncoordinated movements, decreased locomotoractivity, ventro-lateral recumbency, quick breathing, labored respiration rates, shallow respiration, swelling of the genital region and abdomen, piloerection, red discoloration of urine, diarrhea, salivation, chromodacryorrhea of both eyes and snout, lean appearance, and ptosis.Salivation and diarrhea were also observed at the 150 mg/kg/day dose level.Body weights and body weight gain of 1000 mg/kg/day-treated males were statistically significantly decreased during treatment. Females treated at 1000 mg/kg/day had statistically significantly decreased body weights and body weight gain during the pre-mating period.These females also had decreased body weights during pregnancy and lactation.No abnormal body weight changes among rats treated at 50 or 150mg/kg/day.

Increased severity of hematopoiesis in the spleen was observed in 1000 mg/kg/day-treated males. Increased severity of hemosiderosis in the spleen was observed in 1000 mg/kg/day-treated females. Males in the 1000 mg/kg/day dose group had increased relative spleen weights.The following hematologyp arameters were statistically increased among 1000 mg/kg/day-treated males and females: erythrocytes count, hemoglobin concentration, and hematocrit. In addition, these males had statistically significantly increased: mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobinconcentration, and red cell distribution width. Females in the 1000 mg/kg/day dose group had statistically significantly decreased neutrophils count. No toxicologically relevant changes in organ weight or histopathology of the thymus. Minimal to slight atrophy in the thymus (one male and one female of the1000 mg/kg/day dose group) was considered to be due to inanition or stress, rather than direct effects of the compound.

Males in the 1000 mg/kg/day dose group had increased relative liver weights, and females in the same dose group had increased absolute and relative liver weights. Minimal to slight hepatocyte hypertrophy was observed in 1000 mg/kg/day-treated males and females. Males in the 1000 mg/kg/day dose group had increased levels of alanine aminotrasferase, alkaline phosphatase, and triglycerides.Females in the 1000 mg/kg/day dose group had decreased levels of aspartate aminotransferase and bilirubin and increased albumin levels.

Meningitis was present in the brain and spinal cord of two females in the 1000 mg/kg/day dose group. These deaths are likely to be incidental findings; however, a possible relationship to treatment could not be entirely excluded. Lethargy, uncoordinated movements, and decreased locomotor activity were observed in the 1000 mg/kg/day dose group.There were no changes in static righting reflex and grip strength in test article-treated animals when compared to control group animals.

Males treated at 1000 mg/kg/day had increased relative epididymides weights. During necropsy, 4/10 males in the 1000 mg/kg/day dose group appeared tohave yellowish nodule(s) on the epididymides. This finding correlated with the microscopic finding of slight to moderate sperm granuloma and was considered to be treatment-related.Other findings in the testes (tubular atrophy, dilation, giant cells) and epididymides (reduced spermatozoa, cellular debris) of 1000mg/kg/day treated males were secondary to the blockage caused by the sperm granulomas. There was no evidence of impaired spermatogenesis. All reproductive parameters among animals treated at 50 or 150 mg/kg/day were normal.

All females mating within 4 days of pairing. In the 1000 mg/kg/day dose group, 4 pregnancies occurred out of 9 mated females.One of these pregnant females died spontaneously on day 12 post-coitum; at necropsy, this female showed implantation sites.As a result, only 3 litters with living pups were recorded in thisdose group.This gave rise to a decreased fertility index, conception rate, and gestation index.

Of 25 pups in 3 litters, at first litter check, 12 died (across 2 litters) by day 4. Mean body weights of pups per group in the 1000 mg/kg/day dose group were statistically significantly decreased on Days 1 and 4 during lactation. Clinical signs (small, cold or pale appearance, and little or no milk) were observed in all groups, but the incidence in the 1000 mg/kg/day group was slightly increased.Macroscopic examination of pups revealed small appearance, no milk, and cannibalism.The incidence of small pups was increased in the 1000 mg/kg/day group, which corresponded with the observed decreased body weights in this group.

Based on the results of the study, the No Observed Adverse Effect Level for the test article is 150 mg/kg/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
Remarks:
No deviations ocurred that negatively impacted the integrity of the study.
GLP compliance:
yes
Limit test:
no
Justification for study design:
Per OECD 422.

Test material

Constituent 1
Chemical structure
Reference substance name:
1-BUTANESULFONAMIDE, 1,1,2,2,3,3,4,4,4-NONAFLUORO-N-METHYL-
EC Number:
614-396-3
Cas Number:
68298-12-4
Molecular formula:
C5H4F9NO2S
IUPAC Name:
1-BUTANESULFONAMIDE, 1,1,2,2,3,3,4,4,4-NONAFLUORO-N-METHYL-
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M Company, Lot 3
- Expiration date of the lot/batch: 17 June, 2004
- Purity test date:
30 January, 2002
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature in the dark.
- Stability under storage conditions:
Stable
- Stability under test conditions:
Stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
The test article was tested as a suspension in propylene glycol and was determined to be stable for
at least 4 hours at room temperature.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test article was suspended in propylene glycol.
- Final preparation of a solid:
Suspended in propylene glycol.
FORM AS APPLIED IN THE TEST: The test article was suspended in propylene glycol

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species and strain commonly used in reproductive and developmental screening studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland
- Females (if applicable) nulliparous and non-pregnant:Yes
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: Male mean: 250-258 grams, Female mean: 183-186 grams.
- Fasting period before study: None
- Housing: 5 animals per sex per cage. During mating, females were caged together with males on
a one-to-one basis. Mated females and males were individually housed in labelled polycarbonate
cages containing sawdust as bedding.
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet.
- Water (e.g. ad libitum): Tap water ad libitum.
- Acclimation period: 5 days.
DETAILS OF FOOD AND WATER QUALITY: Food was analyzed for nutriets and contaminants on a
regular basis. Tap water was analyzed quarterly.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1-23.5 C.
- Humidity (%): 42-87 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 21 July, 2003 To: 05 September, 2003

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared daily within 4 hours prior to
dosing and were homogenized to visually acceptable levels. Adjustment was made for specific gravi
ty of the test substance and vehicle.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test article solubility and test
system compatibility.
- Concentration in vehicle: To appropriately dose 50, 150 and 1000 mg/kg/day at a maximum dose
volume of 5 mL/kg body weight.
- Amount of vehicle (if gavage): 5 mL/kg body weight.
- Lot/batch no. (if required): No data
- Purity: No data.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: No data
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): Individually
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
The males and females were exposed for 2 weeks prior to mating, aduring mating and up to the day prior to necropsy. Exposure period was at least until the minumum total dosing period of 28 days had been completed.
Frequency of treatment:
Once daily
Details on study schedule:
Screening study.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control (Propylene glycol)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 3
No. of animals per sex per dose:
10 per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a range-finding study in which dose levels of 150
and 1000 mg/kg were administered for 5 day.
- Rationale for animal assignment (if not random): Random
- Fasting period before blood sampling for clinical biochemistry: Overnight
Positive control:
None

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily
BODY WEIGHT: Yes
- Time schedule for examinations: weekly; Days 1 and 4 of lactation
FOOD EFFICIENCY: Weekly
HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to necropsy.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight.
- How many animals: 5 per sex
- Parameters checked:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Platelet count
Red cell distribution
Total leucocyte count
Differential leucocyte count
Prothrombin time
Partial thromboplastin time
CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
immediately prior to necropsy.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight.
- How many animals:
5 per sex
- Parameters checked:
Alanine aminotransferase
Alkaline phophatase
Aspartate aminotransferase
Bilirubin, total
Chloride
Cholesterol, total
Creatinine
Glucose
Phosphorous
Protein, total
Protein, albumin
Triglycerides
Urea
Calcium
Potassium
Sodium
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to necropsy.
- Dose groups that were examined: 5 per sex per group.
- Battery of functions tested: sensory activity, grip strength, motor activity
IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
No data.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in offspring:
- The numbers of live and dead pups at the First Litter Check (check at day 1 of lactation) and daily thereafter.
- The individual weight of all live pups on days 1 and 4 of lactation.
- Sex of all pups (by assessment of ano-genital distance).
- The number of pups with physical or behavioral abnormalities daily.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities; possible cause of death was determined if possible.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: None

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: None
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after a minimum of 28 days of exposure.
- Maternal animals: All surviving animals were sacrificed at day 4 post partum or shortly thereafter.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
See attached table.
Postmortem examinations (offspring):
SACRIFICE
- Pups were sacrificed on Day 4 of lactation or shortly therafter.
- All pups were sexed and externally examined if possible. The stomach was examined for the presence of milk. Description of all macroscopic abnormalities were recorded.

GROSS NECROPSY
None, external examinations of the pups only.

HISTOPATHOLOGY / ORGAN WEIGTHS
None
Statistics:
If variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
The Steel-test was applied instead of the Dunnett-test if the data could not be assumed to follow a normal distribution.
The exact Fisher-test was applied for 2x2 tables if variables could be dichotomized without loss of information.
All test were two-sided and in all cases p< 0.05 was accepted as the lowest level of significance.
Reproductive indices:
Percentage mating, fertility index, conception rate, gestation index, duration of gestation
Offspring viability indices:
Percentage of live males at First Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss days 0-4 post partum, viability index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, the following clinical observations were noted: lethargy, hunched posture, uncoordinated movements, decreased locomotoractivity, ventro-lateral recumbency, quick breathing, labored respiration rates, shallow respiration, swelling of the genital region and abdomen, piloerection, reddiscoloration of urine, diarrhea, salivation, chromodacryorrhea of both eyes and snout, lean appearance, and ptosis. Salivation and diarrhea were alsoobserved at the 150 mg/kg/day dose level.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
At 1000 mg/kg/day, two females (one found dead, one euthanized in extremis) died due to meningitis and three males (two founddead, one euthanized in extremis) died due to gavage error (1 male) or cause of death not evident (2 males). At 150 mg/kg/day, one female was euthanized inextremis due
to a uterine prolapse just after delivery. All deaths are likely to be incidental findings; however, a possible relationship to treatment cannot entirely be excluded.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of 1000 mg/kg/day-treated males were statistically significantly decreasedduring treatment in 70% of animals. Females treated at 1000 mg/kg/day had statistically significantly decreased body weights and body weight gain during thepre-mating period. These females also had decreased body weights during pregnancy and lactation. No abnormal body weight changes among rats treated at50 or 150 mg/kg/day
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, absolute and relative food consumption were decreased during the complete treatment period. Statistical significance was reached for food consumption and relative food consumption during pre-mating (males), and for food consumption during post-mating (males) and lactation
(females).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant differences of hematology parameters were recorded in the highest dose group at the end of treatment:
- Decreased neutrophil count (females)
- Increased erythrocyte count (males and females)
- increased hemoglobin concentration (males and females)
- Increased hematocrit (males and females)
- Increased mean corpuscular volume (males)
- Increased mean corpuscular hemoglobin (males)
- Increased mean corpuscular hemoglobin concentration (males)
- Increased red cell distribution width (males)
No other statistically significant effects upon hemotology parameters were noted.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant differences in clinical biochemistry parameters were recorded in
the highest dose group at the end of treatment:
- Increased alanine aminotransferase (males)
- Increased alkaline phosphatase (males)
- Increased urea (males)
- Increased chloride (males)
- Increased inorganic phosphorous (males)
- Decreased aspartate aminotransferase (females)
- Decreased bilirubin (females)
- Increased albumin (females)
No other statistically significant effects upon clinical chemistry parameters were noted.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No changes were observed in hearing ability, papillary reflex, static righting reflex and grip strength in the treated animals compared to controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Meningitis was present in the brain and spinal cord of two females in the 1000 mg/kg/day dose group. These deaths are likely to be incidental findings; however, a possible relationship to treatment could not be entirely excluded. Lethargy, uncoordinated movements, and decreased locomotor activity
were observed in the 1000 mg/kg/day dose group. There were no changes in static righting reflex and grip strength in test article treated animals when compared to control group animals.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Primary treatment-related findings were confined to the liver, spleen and epididymides of high dose animals:
Liver: Minimal/slight hepatocyte hypertrophy in males and females
Spleen: Increased severity of hematopoiesis in males and females
Epididymides: Slight/moderate sperm granuloma in males
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No noeplastic lesions were observed upon histopathological examination.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Males treated at 1000 mg/kg/day had increased relative epididymides weights. During necropsy, 4/10 males in the 1000 mg/kg/day dose group appeared to have yellowish nodule(s) on the epididymides. This finding correlated with the microscopic finding of slight to moderate sperm granuloma and was considered to be treatment-related. Other findings in the testes (tubular atrophy, dilation, giant cells) and epididymides (reduced spermatozoa, cellular debris) of 1000 mg/kg/day treated males were secondary to the blockage caused by the sperm granulomas. There was no evidence of impaired spermatogenesis. Al lreproductive parameters among animals treated at 50 or 150 mg/kg/day were normal.
Description (incidence and severity):
All females mating within 4 days of pairing. In the 1000 mg/kg/day dose group, 4 pregnancies occurred out of 9 mated females. One of these pregnant femalesdied spontaneously on day 12 post-coitum; at necropsy, this female showed implantation sites. As a result, only 3 litters with living pups were recorded in this dose group. This gave rise to a decreased fertility index, conception rate, and gestation index.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
neuropathology
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
central nervous system
Organ:
brain
spinal cord
Treatment related:
yes
Dose response relationship:
no
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
other: Epididymides.
Treatment related:
yes
Dose response relationship:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs (small, cold or pale appearance, and little or no milk) were observed in all groups, but the incidence in the 1000 mg/kg/day group was slightly increased.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, of 25 pups in 3 litters, at first litter check, 12 died (across 2 litters) by day 4.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of pups per group in the 1000 mg/kg/day dose group werestatistically significantly decreased on Days 1 and 4 during lactation.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic examination of pups revealed small appearance, no milk, andcannibalism. The incidence of small pups was increased in the 1000 mg/kg/day group, which corresponded with the observed decreased body weights in thisgroup.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the reproductive, developmental and parental systemic No Observed Adverse Effect Level for the test article is 150 mg/kg/day.
Executive summary:

The subacute and reproductive/developmental toxicity of the test article (off white solid, Lot 3) was evaluated in Wistar rats following daily oral gavage during premating, mating, gestation, and lactation. This study was performed in compliance with OECD GLP(1997).The study method was based on OECD 422 (1996).The test article was prepared in propylene glycol (vehicle) just prior to each dosing. Rats (10/sex/dose)received 0 (vehicle; Group 1), 50 (Group 2), 150 (Group 3), or 1000 (Group 4) mg/kg/day test article via oral gavage at a dose volume of 5 mL/kg.The males and females were exposed for 2 weeks prior to mating and during mating. Females were paired 1:1 with males from the same treatment group. Mating was confirmed by copulation plug absence (designated as Gestation Day 0). Males were treated for at least 28 days total.In addition to exposure during prematingand mating, females were exposed during gestation and for at least 4 days of lactation.The offspring were euthanized on Day 4 of lactation or shortly thereafter.The following parameters were evaluated: clinical observations (at least once daily), functional observations (5/sex/group; prior to termination), body weights(weekly; Days 1 and 4 of lactation), food consumption (weekly), reproductive processes, offspring observations, necropsy (termination), clinical chemistry (termination) and organ weights and/or histopathology of select organs (termination). At 1000 mg/kg/day, two females (one found dead, one euthanized in extremis) died due to meningitis and three males (two found dead, one euthanized in extremis) died due to gavage error (1 male) or cause of death not evident (2 males). At 150 mg/kg/day, one female was euthanized in extremis due to a uterine prolapse just after delivery. All deaths are likely to be incidental findings; however, a possible relationship to treatment cannot entirely be excluded.At 1000 mg/kg/day, the following clinical observations were noted: lethargy, hunched posture, uncoordinated movements, decreased locomotoractivity, ventro-lateral recumbency, quick breathing, labored respiration rates, shallow respiration, swelling of the genital region and abdomen, piloerection, red discoloration of urine, diarrhea, salivation, chromodacryorrhea of both eyes and snout, lean appearance, and ptosis.Salivation and diarrhea were also observed at the 150 mg/kg/day dose level.Body weights and body weight gain of 1000 mg/kg/day-treated males were statistically significantly decreased during treatment. Females treated at 1000 mg/kg/day had statistically significantly decreased body weights and body weight gain during the pre-mating period.These females also had decreased body weights during pregnancy and lactation.No abnormal body weight changes among rats treated at 50 or 150mg/kg/day.

Increased severity of hematopoiesis in the spleen was observed in 1000 mg/kg/day-treated males.Increased severity of hemosiderosis in the spleen was observed in 1000 mg/kg/day-treated females. Males in the 1000 mg/kg/day dose group had increased relative spleen weights.The following hematologyp arameters were statistically increased among 1000 mg/kg/day-treated males and females: erythrocytes count, hemoglobin concentration, and hematocrit. In addition, these males had statistically significantly increased: mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobinconcentration, and red cell distribution width. Females in the 1000 mg/kg/day dose group had statistically significantly decreased neutrophils count. No toxicologically relevant changes in organ weight or histopathology of the thymus. Minimal to slight atrophy in the thymus (one male and one female of the1000 mg/kg/day dose group) was considered to be due to inanition or stress, rather than direct effects of the compound.

Males in the 1000 mg/kg/day dose group had increased relative liver weights, and females in the same dose group had increased absolute and relative liverweights. Minimal to slight hepatocyte hypertrophy was observed in 1000 mg/kg/day-treated males and females. Males in the 1000 mg/kg/day dose group had increased levels of alanine aminotrasferase, alkaline phosphatase, and triglycerides.Females in the 1000 mg/kg/day dose group had decreased levels of aspartate aminotransferase and bilirubin and increased albumin levels.

Meningitis was present in the brain and spinal cord of two females in the 1000 mg/kg/day dose group. These deaths are likely to be incidental findings;however, a possible relationship to treatment could not be entirely excluded. Lethargy, uncoordinated movements, and decreased locomotor activity were observed in the 1000 mg/kg/day dose group.There were no changes in static righting reflex and grip strength in test article-treated animals when compared to control group animals.

Males treated at 1000 mg/kg/day had increased relative epididymides weights.During necropsy, 4/10 males in the 1000 mg/kg/day dose group appeared tohave yellowish nodule(s) on the epididymides. This finding correlated with the microscopic finding of slight to moderate sperm granuloma and was considered to be treatment-related.Other findings in the testes (tubular atrophy, dilation, giant cells) and epididymides (reduced spermatozoa, cellular debris) of 1000mg/kg/day treated males were secondary to the blockage caused by the sperm granulomas. There was no evidence of impaired spermatogenesis. All reproductive parameters among animals treated at 50 or 150 mg/kg/day were normal.

All females mating within 4 days of pairing.In the 1000 mg/kg/day dose group, 4 pregnancies occurred out of 9 mated females.One of these pregnant females died spontaneously on day 12 post-coitum; at necropsy, this female showed implantation sites.As a result, only 3 litters with living pups were recorded in thisdose group.This gave rise to a decreased fertility index, conception rate, and gestation index.

Of 25 pups in 3 litters, at first litter check, 12 died (across 2 litters) by day 4.Mean body weights of pups per group in the 1000 mg/kg/day dose group were statistically significantly decreased on Days 1 and 4 during lactation. Clinical signs (small, cold or pale appearance, and little or no milk) were observed in all groups, but the incidence in the 1000 mg/kg/day group was slightly increased.Macroscopic examination of pups revealed small appearance, no milk, and cannibalism.The incidence of small pups was increased in the 1000 mg/kg/day group, which corresponded with the observed decreased body weights in this group.

Based on the results of the study, the reproductive, developmental and parental systemic No Observed Adverse Effect Level for the test article is 150 mg/kg/day.

Categories Display