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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

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)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-benzyl-3-carboxylatopyridinium sodium chloride
EC Number:
268-692-9
EC Name:
1-benzyl-3-carboxylatopyridinium sodium chloride
Cas Number:
68133-60-8
Molecular formula:
C13H12NO2.Cl.Na
IUPAC Name:
1-benzyl-3-carboxylatopyridinium sodium chloride
Details on test material:
- Name of test material (as cited in study report): LUGALVAN BPC dry
- Physical state: solid
- Analytical purity: 95.5 area-%
- Lot/batch No.: 467602
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: body weight of all animals within ± 20% of the sex mean.
- Housing:
Pre-mating:
Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating:
Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating:
Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation:
Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the vehicle was chosen based on trial formulations performed at WIL Research Europe
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group,
avoiding sibling mating. Mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory
plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was
allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Detection of mating was not confirmed for animal nos. 47 and 57 which did deliver live offspring. The mating date of this animal was estimated at
21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5
hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands.
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 44-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
once daily
Details on study schedule:
N/A
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of the 14-Day dose range finding study
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Positive control:
N/A

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
At least twice daily for mortality. Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were conducted for all animals, at least immediately after dosing (based on results of the dose range finding study, Project 501951; BASF Project 01R0642/12X355, where no clinical signs were observed). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was
predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs,
only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

General reproduction data
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of
pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was
examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest,
pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Oestrous cyclicity (parental animals):
N/A
Sperm parameters (parental animals):
Of all animals of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
Litter observations:
Each litter was examined to determine the following, if practically possible:

Mortality / Viability:
The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.

Clinical signs:
At least once daily, detailed clinical observations were made for all animals.

Body weights:
Live pups were weighed on Days 1 and 4 of lactation.

Sex
Sex was determined for all pups on Days 1 and 4 of lactation.

Necropsy pups
Pups surviving to planned termination were killed by decapitation on Days 5-6 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
All males and the selected 5 females/group were deprived of food overnight, the evening before the scheduled necropsy (with a maximum of 24 hours), but water was provided. Non-selected females were not deprived of food.
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
Necropsy was conducted on the following days:
Females which delivered on Lactation Days 5-8.
Females which failed to deliver on Post-coitum Days 27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
Males: Following completion of the mating period (a minimum of 28 days of dose administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The numbers of former implantation sites and corpora lutea were recorded for all paired females. These numbers were not reported for non-pregnant and non-mated females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at WIL Research Europe using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches [jejunum, ileum] if detectable, Brain - cerebellum, mid-brain, cortex, Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Coagulation gland, Sciatic nerve Duodenum, Seminal vesicles, Epididymides[1], Skeletal muscle, Eyes (with optic nerve (if detectable) and Harderian gland)[1], (Skin), Spinal cord -cervical, midthoracic, lumbar, (Male and female mammary gland area), Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach (forestomach and glandular stomach), Ileum, Testes[1], Jejunumm, Thymus, Kidneys, Thyroid including parathyroid if detectable, (Lacrimal gland, exorbital), (Tongue), (Larynx), Trachea, Liver, Urinary bladder, Lung, infused with formalin, Uterus, Lymph nodes - mandibular, mesenteric, Vagina, (Nasopharynx), All gross lesions, (Esophagus)

[1]: Fixed in modified Davidson's solution (prepared at WIL Research Europe using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

HISTOPATHOLOGY: Yes
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Of all animals of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
A peer review on the histopathology data was performed by a second pathologist.

The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of all group 1 and 4 animals and of all males that failed to sire and all females that failed to deliver healthy pups
[*] Reproductive organs includes the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

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


ORGAN WEIGHTS
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group (see Allocation):
Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate[2], Liver, Seminal vesicles including coagulating glands[2], Ovaries, Thyroid including parathyroid[2],
[2] weighed when fixed for at least 24 hours.
All remaining males:
Epididymides and Testes
Postmortem examinations (offspring):
Necropsy pups
Pups surviving to planned termination were killed by decapitation on Days 5-8 of lactation.

Pups found dead during the weekend were fixed in identified containers containing 96% ethanol (in glycerine, both from Klinipath, Duiven, The Netherlands).
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used: Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%), Fertility index (%), Conception index (%), Gestation index (%), Duration of gestation
Offspring viability indices:
For each group, the following calculations were performed: Percentage live males at First Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss Days 0-4 of lactation, Viability index

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred during the study period.

No clinical signs of toxicity were noted during the observation period.

Salivation was noted for one male at 1000 mg/kg bw/day and alopecia was noted for two control females. These were incidental in nature and were not toxicologically relevant.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No toxicologically relevant changes in body weights and body weight gain were noted.

Body weights were significantly higher for females at 300 mg/kg bw/day from lactation Days 1-4. The differences from controls was slight, occurred in the absence of a dose related trend, and was therefore considered to be a chance finding.

Food consumption before or after allowance for body weight was similar between treated and control animals.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects were observed concerning staging of spermatogenesis

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically relevant effects on reproductive parameters were noted.

The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

For females 44 (Control) and 70 (300 mg/kg bw/day), the number of pups were slightly higher than the number of implantations. This was considered to be caused by normal resorption of these areas as these enumerations were performed on Day 5 of lactation.

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Gestation
The gestation index and duration of gestation were unaffected by treatment up to 1000 mg/kg bw/day.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Absolute and relative liver weights were significantly higher for females at 300 and 1000 mg/kg bw/day.

In the absence of any toxicologically relevant effects on the liver noted at the microscopic examination, the higher liver weights were not considered to be toxicologically relevant. Terminal body weights were significantly higher for females at 300 mg/kg bw/day and relative adrenal
weights were significantly increased for males at this dose level as well. In the absence of dose-dependency, these differences were not considered
to be toxicologically relevant.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Incidental findings noted for control and/or treated animals included thickened wall of the duodenum or jejunum, nodules, enlarged or reduced size of the epididymides, testes, lymph nodes, or liver, flaccid testes, pelvic dilation of the kidney, ectopic splenic tissue, discoloration of or focus/foci on the lymph nodes, stomach glandular mucosa, clitoral glands, lungs, thymus, or adrenal glands and alopecia. These findings remained within the background range encountered for animals of this age and strain and were not considered to be toxicologically relevant.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related microscopic findings.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of test item related impaired spermatogenesis. No histopathological changes were noted in the reproductive organs of the animals that failed to conceive or deliver healthy offspring that could account for failing to deliver healthy offspring.
There was one 300 mg/kg bw/day (Group 3) treated animal (male 21) with bilateral complete seminiferous tubular atrophy in the testes. No recognizable stages were present in the testes of this male rat.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item. All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
parental, reproduction and developmental
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Generation: P and F1 (migrated information)

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
One pup at 100 mg/kg bw/day and four pups at 1000 mg/kg bw/day died or went missing in the first days of lactation. No pups in the control or 300 mg/kg bw/day groups died or went missing. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
Incidental clinical symptoms of pups consisted of scabs on the mouth, blue spot on the head, reduced size of the ear and lean appearance. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment.

Early postnatal pup development
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Macroscopy
Incidental macroscopic findings of pups that were found dead included beginning autolysis. Incidental macroscopic findings among surviving pups included lean appearance and scab on the mouth. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

LUGALVAN BPC dry was administered by daily oral gavage to male and female Wistar Han rats at

dose levels of 100, 300 and 1000 mg/kg. Males were exposed for 2 weeks prior to mating, during

mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating,

during mating, during post-coitum, and at least 4 days of lactation (for 44-49 days).

Formulation analysis showed that the formulations were prepared accurately, were homogenous, and

were stable for at least 5 hours at room temperature.

Parental results:

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day).

Reproductive results:

No reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day).

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day).

In conclusion, treatment with LUGALVAN BPC dry by oral gavage in male and female Wistar Han rats

at dose levels of 100, 300 and 1000 mg/kg bw/day revealed no parental, reproductive or

developmental toxicity up to 1000 mg/kg bw/day. Based on these results, a parental, reproduction and

developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was

derived.

Applicant's summary and conclusion