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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: screening test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-12-07 to 2012-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012
Reference Type:
other: Appendix 3 of NOTOX Project 498332
Title:
Unnamed
Year:
2012

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: White solidified mass or clear colourless liquid depending on ambient temperature
Details on test material:
Please refer to the section "confidential details on test material" below.

Test animals

Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
- Source F0: Charles River Deutschland, Sulzfeld, Germany
- Age at start F0-treatment: Approximately 11 weeks old
- Weight at study initiation:
# Pre-Mating males (group 1/2/3/4): 311/310/312/310 g (means)
# Pre-Mating females (group 1/2/3/4): 196/193/196/197 g (means)
- Fasting period before study: No data
- 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.
# General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water
- Acclimation period F0: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 3°C
- Humidity: 40 - 70%
(Cleaning procedures in the room might have caused the temporary fluctuations above the optimal
maximum level of 70% for relative humidity (with a maximum of 4 hours)).
- Air changes: 15 room air changes/hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 2011-12-11 To: 2012-02-02

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036 (Merck, Darmstadt, Germany)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. The test substance was heated before weighing (maximum temperature=49.7°C, maximum duration=4 hours and 20 minutes). Adjustment was made for the specific gravity of the vehicle and the relative density of the test substance.

VEHICLE
- Justification for use and choice of vehicle: Propylene glycol, standard vehicle for studies of this type
- Concentration in vehicle: 1.2, 6, 30 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw (actual dose volumes were calculated according to the latest body weight)
- Lot/batch no.: No data
- Purity: No data
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: mating period of 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually


Mating procedures:
one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of 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.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method (NOTOX Project 498338, BASF Project 05Y0436/11X294). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 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 43 - 53 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). Female nos. 44 (Group 1 - vehicle control), 51 (Group 2 - 6 mg/kg bw/d) and 72 (Group 4 - 100 mg/kg bw/d) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose administration.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
6 mg/kg bw/d
Basis:
analytical conc.
low-dose level
Remarks:
Doses / Concentrations:
30 mg/kg bw/d
Basis:
analytical conc.
mid-dose level
Remarks:
Doses / Concentrations:
150 mg/kg bw/d
Basis:
analytical conc.
high-dose level
No. of animals per sex per dose:
10 animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on results of a 14-day pilot study (NOTOX Project 498335; BASF Project 01R0436/11X204) using dose levels of 30, 100 and 300 mg/kg bw/d, and in consultation with the Sponsor. At 300 mg/kg bw/d, the most common clinical signs included uncoordinated movements and flat posture, with other clinical signs included lethargy, hunched posture, laboured respiration, rales, salivation, and piloerection noted only on 1-2 occasions. Uncoordinated movements were also commonly noted for animals at 100 mg/kg bw/d. In both cases, the clinical signs were transient, resolving by 4 hours post-dosing. No clinical signs were noted for animals at 30 mg/kg bw/d. Body weight gains were lower at 300 mg/kg bw/d in males. There were no effects on food consumption, clinical pathology, macroscopic findings or organ weights at any dose level.

- Rationale for animal assignment (F0):
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:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily (including observations for Mortality/Viability)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards

BODY WEIGHT: Yes
- Time schedule for examinations: 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: yes
- 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 postcoitum and on Days 1 and 4 of lactation.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (deprived of food overnight, with a maximum of 20 hours)
- How many animals: selected 5 animals/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before necropsy
- Animals fasted: Yes (deprived of food overnight, with a maximum of 20 hours)
- How many animals: selected 5 animals/sex/group

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling).
- Dose groups that were examined: selected 5 animals/sex/group
- Battery of functions tested: hearing ability / pupillary reflex / static righting reflex / grip strength / locomotor activity

Sperm parameters (parental animals):
Parameters examined in F0 male parental generation:
- testis weight, epididymis weight
other: Histopathologic examination was performed on the reproductive organs from 5 selected males and females of Group 1 (control) and Group 4 (150 mg/kg bw/day), as well as all organs with macroscopic findings from all rats (i.e. testis). Of the all Group 1 and 4 males and of the males suspected to be infertile, additional slides of 3-4 micrometers of the testes were prepared and stained with PAS/hematoxylin to examine staging of spermatogenesis.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring (each litter):
- 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.
Postmortem examinations (parental animals):
SACRIFICE
All males and the selected 5 females/group were deprived of food overnight (with a maximum of approximately 20 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.
Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane and subsequently exsanguinated.
- Male animals: All surviving animals, following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: All surviving animals, females which delivered: Lactation Days 6-7 and females which failed to deliver* (nos. 57, 60, 63, 67 and 74): Post-coitum Days 25-28 (females with evidence of mating)
*) 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.


GROSS NECROPSY
- Gross necropsy consisted of 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.


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
- Adrenal glands
- Brain
- Epididymides
- Heart
- Kidneys
- Liver
- Ovaries
- Spleen
- Testes
- Thymus
- Uterus (including cervix)
- Prostate
- Seminal vesicles including coagulating glands
- Thyroid including parathyroid

All remaining males:
- Epididymides
- Testes


HISTOPATHOLOGY
All organ and tissue samples, as defined below, were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
Of the all males of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine stages of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.

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 all males of Groups 1 and 4 and all males suspected to be infertile to examine stages of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of all animals of Groups 1 and 4, and all males that failed to sire and all females that failed to deliver healthy pups:
Group 1: --
Group 2: Female/Male nos. 51/17, 60/20: No offspring
Group 3: Female/Male nos. 63/23, 67/27: No offspring
Group 4: Female/Male nos. 74/34: No offspring

*) Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 6-7 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination).

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations (stomach)]

NECROPSY PUPS
Pups surviving to planned termination were killed by decapitation on Days 6-7 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.
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:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Fertility index (%) = (Number of pregnant females/Number of females paired) x 100
Conception index (%) = (Number of pregnant females/Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Offspring viability indices:
Percentage live males at First Litter Check = (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check = (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage of postnatal loss Days 0-4 of lactation = (Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check) x 100
Viability index = (Number of live pups on Day 4 post partum/Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred during the study period.
Uncoordinated movements were noted for all animals at 150 mg/kg bw/d through most of the treatment period and flat posture was also noted for all females and 7/10 males. Hunched posture was also commonly noted at this dose level, especially for females, and lethargy, rales and piloerection were noted for only a few animals on limited occasions.
Hunched posture was noted for two females at 6 mg/kg bw/d, though this was not considered to be toxicologically relevant due to the limited incidence and severity noted. Alopecia was noted for one female each at 6 and 30 mg/kg bw/d; this finding was incidental. No clinical signs were noted for control animals of either sex, nor were any signs seen for males at 6 and 30 mg/kg bw/d.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No toxicologically relevant changes in body weights and body weight gain were noted. Females at 6 mg/kg bw/d had significantly higher absolute body weights on lactation Day 1 and lower gains on Day 4. The differences from controls were only slight, occurred in the absence of a dose related distribution, and were not considered treatment related or toxicologically relevant.
Food consumption before or after allowance for body weight was similar between treated and control animals up to 150 mg/kg bw/d. No toxicologically relevant changes were noted in food consumption.

REPRODUCTIVE FINDINGS (PARENTAL ANIMALS)
- Reproduction/developmental data: 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.
- Developmental data: No toxicologically relevant effects on the gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios up to 150 mg/kg bw/d. Statistically significant changes between brain to body weight ratios of females at 6 mg/kg bw/day and control animals were not considered to be a sign of toxicity as no dose-dependency was seen and there were no corroborative findings noted at the microscopic examination.

GROSS PATHOLOGY / HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related microscopic findings; all microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. Furthermore, there were no morphological findings in the reproductive organs of either sex which could be attributed to the test substance.

OTHER: NEUROBEHAVIOUR (PARENTAL ANIMALS)
One male and three females at 150 mg/kg bw/d did not have sufficient grip strength in the functional observational test. This was likely related to the uncoordinated movements seen during clinical observations, which could be indicative of a slight decrement in muscular strength. There were no toxicologically relevant effects on hearing ability, pupillary reflex, or static righting reflex up to 150 mg/kg bw/d.
The variation in motor activity did not indicate a relation with treatment. Males and females at 150 mg/kg bw/d had lower activity compared to controls in the first few blocks of testing, which was likely related to the movement-related clinical signs at this dose level. These signs were transient. Females at 6 mg/kg bw/d had significantly higher basic movements and ambulatory counts, however, the differences from controls were only slight and in the absence of a treatment related distribution, were not considered treatment-related or toxicologically relevant. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period. Males at 150 mg/kg bw/d, had a somewhat blunted habituation effect compared to controls, though they still habituated over the test session. This was also related to the clinical signs noted, as was not considered adverse.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(parental toxicity)
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on clinical signs of systemic toxicity noted at 150 mg/kg bw/d
Dose descriptor:
NOAEL
Remarks:
(developmental toxicity)
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effects on developmental parameters

Results: F1 generation

Details on results (F1)

GENERAL (OFFSPRING)
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 macroscopic examination did not reveal treatment-related findings.

VIABILITY (OFFSPRING)
Three pups of the 6 and 150 mg/kg bw/d groups were found dead or went missing during the first days of lactation. Missing pups were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
Incidental clinical symptoms of pups consisted of no milk in the stomach, pale or lean appearance and missing tail. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment up to 150 mg/kg bw/d.

SEXUAL MATURATION (OFFSPRING)
--

ORGAN WEIGHTS (OFFSPRING)
Not determined.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings of pups that were found dead included absence of milk in the stomach, insufficient milk in the stomach, cannibalism of the abdominal organs. Missing tail was the only finding noted for surviving pups, which was incidental in nature. The type and frequency of these findings remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

HISTOPATHOLOGY (OFFSPRING)
--

OTHER FINDINGS (OFFSPRING)
--

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effects on developmental parameters.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

All rats were necropsied. Histopathologic examination was performed on an extensive list of organs and tissues from 5 selected males and females of Group 1 (control) and Group 4 (150 mg/kg bw/day), the reproductive organs from all Group 1 and Group 4 rats, as well as all organs with macroscopic findings from all rats. Of the all Group 1 and 4 males and of the males suspected to be infertile, additional slides of 3-4 micrometers of the testes were prepared and stained with PAS/hematoxylin to examine staging of spermatogenesis. There were no unscheduled deaths. There were no treatment-related macroscopic findings. There were no treatment-related microscopic findings. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test substance.

Applicant's summary and conclusion