Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 December 2008 - 26 August 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations.
Qualifier:
according to
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Molecular formula: C10H26Cl2N2O2 (component A), C14H34Cl2N2O3 (component B)
- Molecular weight: 277.23 (component A), 349.34 (component B)
- Physical state: Clear yellow / orange liquid
- Analytical purity: ~ 100%
- Composition of test material, percentage of components: This product consisted essentially of two components, derived from the reaction of ethylene glycol and butylene oxide in ratios of 2/1 and 3/1. The 2/1 adduct (component A) was approximately 80% of the mixture and the 3/1 adduct (component B) was approximately 20%.
- Lot/batch No.: first batch 8666-080-C, second batch 8470-004-A, third batch 8740-054-C
- Expiration date of the lot/batch: first batch 15 June 2009, second batch 15 November 2009, third batch 11 May 2010 (allocated by NOTOX, 1 year after receipt of the test substance)
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark
- Other: hygroscopic.
- Until 13 March 2009 batch 8666-080-C was used. The concentration of the salt in this solution was 70.5%, pH 7.2, density 1.106 g/ml at 25°C
- From 13 March 2009 until 03 June 2009 batch 8470-004-A was used. The salt of this batch was made from the same amine as used for preparation of the previous batch (8666-080-C). Consequently, the chemical composition of the two batches should be identical. The concentration of the salt in this solution was 62%. pH 7.1, density 1.1037 g/ml at 25°C
- From 03 June 2009 onwards batch 8740-054-C was used. The salt of this batch was made from a different precursor diamine as used for preparation of the previous two batches (8666-080-C and 8740-004-A). However, the amine content of the precursor diamine (batch 8740-054-A) used to make batch 8740-054-C of the XTJ-568 dihydrochloride salt had a value of 8.72 meq/g which is very close to the 8.84 meq/g value of the precursor diamine used earlier (batch 8376-032-H). The difference of approximately 1.4% fell within the normal variation of the amination process used to prepare XTJ-568. Therefore, the chemical composition of the three batches was comparable. The concentration of the salt in this solution was 63.73%. pH 7.2, density 1.1014 at 25°C
Specific details on test material used for the study:
- Molecular formula: C10H26Cl2N2O2 (component A), C14H34Cl2N2O3 (component B)
- Molecular weight: 277.23 (component A), 349.34 (component B)
- Physical state: Clear yellow / orange liquid
- Analytical purity: ~ 100%
- Composition of test material, percentage of components: This product consisted essentially of two components, derived from the reaction of ethylene glycol and butylene oxide in ratios of 2/1 and 3/1. The 2/1 adduct (component A) was approximately 80% of the mixture and the 3/1 adduct (component B) was approximately 20%.
- Lot/batch No.: first batch 8666-080-C, second batch 8470-004-A, third batch 8740-054-C
- Expiration date of the lot/batch: first batch 15 June 2009, second batch 15 November 2009, third batch 11 May 2010 (allocated by NOTOX, 1 year after receipt of the test substance)
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark
- Other:
- hygroscopic.
- Until 13 March 2009 batch 8666-080-C was used. The concentration of the salt in this solution was 70.5%, pH 7.2, density 1.106 g/ml at 25°C
- From 13 March 2009 until 03 June 2009 batch 8470-004-A was used. The salt of this batch was made from the same amine as used for preparation of the previous batch (8666-080-C). Consequently, the chemical composition of the two batches should be identical. The concentration of the salt in this solution was 62%. pH 7.1, density 1.1037 g/ml at 25°C
- From 03 June 2009 onwards batch 8740-054-C was used. The salt of this batch was made from a different precursor diamine as used for preparation of the previous two batches (8666-080-C and 8740-004-A). However, the amine content of the precursor diamine (batch 8740-054-A) used to make batch 8740-054-C of the XTJ-568 dihydrochloride salt had a value of 8.72 meq/g which is very close to the 8.84 meq/g value of the precursor diamine used earlier (batch 8376-032-H). The difference of approximately 1.4% fell within the normal variation of the amination process used to prepare XTJ-568. Therefore, the chemical composition of the three batches was comparable. The concentration of the salt in this solution was 63.73%. pH 7.2, density 1.1014 at 25°C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: (P=F0) 5-6 wks; (F1) 5-7 wks
- Weight at study initiation: (P=F0) Males: 146-175 g; Females: 109-140 g; (F1) Males: 56-182 g; Females: 48-141 g
- Fasting period before study: not applicable
- Accommodation
Pre-mating: Animals were housed in groups of 4 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 4 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Offspring was kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet: ad libitum, Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: ad libitum, Free access to tap-water.
- Acclimation period F0: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.3 - 22.7°C
- Humidity (%): 21 - 94%
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day.

IN-LIFE DATES: From: 04 December 2008 To: 26 August 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was an amine salt. Since the different batches XTJ-568 dihydrochloride used in this study had different amine salt concentrations, a conversion factor was applied for calculating the amount of test substance needed for formulation. As a result, the prepared formulations contained the same amount of amine salt (= active ingredient) throughout the study:

batch - amine salt concentration (%) - conversion factor
first batch (8666-080-C) - 70.5% - 1.00
second batch (8470-004-A) - 62% - 1.14
third batch (8740-054-C) - 63.73% - 1.11

Vehicle: Water (Elix, Millipore S.A.S., Molsheim, France)
Rationale for vehicle: Based on information provided by the Sponsor and trial formulations performed at NOTOX.

Before use, the container with the test substance was shaken to obtain homogeneity. Formulations (w/w) were prepared daily within 6 hours prior to dosing until January 11th 2009. From January 12th 2009 onwards formulations were prepared weekly based on stability information. The formulations were homogenized to a visually acceptable level. Adjustment was made for density of the test substance.
Details on mating procedure:
Following a minimum of 70 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates for the F0-generation. For the F1-generation parentage was known through mating records (maintained in the raw data). 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 had occurred, the males and females were separated.

A maximum of 15 days was allowed for mating. If no evidence of copulation was obtained after 10 days, the female was placed with another male (for an additional five days) of the same treatment group who had successfully mated, again avoiding sibling mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were done according to a validated method (NOTOX Project 489272) as specified below:
Week 1
Group - Analysis (type of sample)
1 - acc (M)
2 - acc + hom + stabt=0 (TMB) + stabt=6 hours, RT (S) + stabt=8 days, RF (S)
3 - acc (M)
4 - acc + hom + stabt=0 (TMB) + stabt=6 hours, RT (S) + stabt=8 days, RF (S)

Weeks 8, 16 and 33
Group - Analysis (type of sample)
1 - acc (M)
2 - acc + hom (TMB)
3 - acc (M)
4 - acc + hom (TMB)

Duplicate samples were analysed
acc=accuracy, hom=homogeneity, stab=stability
T=top, M=middle, B=bottom position of container
S=stability sample taken at middle position of container
RF=refrigerator, RT=room temperature

The accuracy of preparation was considered acceptable if the mean measured concentrations were 80% - 120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was <= 10%. Formulations were considered stable if the decrease in concentration between the stored and freshly taken samples was less than 10%.

RESULTS:

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).
A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation prepared for use in Week 8 and 16. It was not considered to derive from the formulation since the response was only observed in one of the duplicate samples. The blank response was probably caused by the analytical method used. Due to the nature of the test substance, an LC-MS method without a column (i.e. separation) was used. The absence of a separation was probably the cause for the blank interference, but for this test substance it was considered the best possible. In all other formulations of Group 1, no response at the retention time of the test substance was observed
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation <= 10%).
Formulations were stable during storage at room temperature for at least 6 hours and in a refrigerator for at least 8 days (i.e. a decrease in concentration <10%). Note that some of the formulation samples showed an apparent increase in concentration, but since this was within the +/- 20% limits, it was considered not significant.
Duration of treatment / exposure:
F0-generation: A minimum of 70 days prior to mating and continuing until euthanasia (except for half of F0-males, which were dosed up to three days (Day 89 of the study) prior to scheduled necropsy (Day 92 of the study).
F1-generation (F0-offspring): The F1-generation was potentially exposed to the test substance in utero, through nursing during lactation and directly following weaning. After weaning, pups were treated for a minimum of 70 days prior to mating and continuing until euthanasia.
F2-generation (F1-offspring): The F2-generation was potentially exposed to the test substance in utero and through nursing during lactation.
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. Animals were dosed up to the day prior to scheduled necropsy, except for half of F0-males. Due to shortage in test substance, the latter group of animals was dosed up to three days (i.e. Day 89 of the study) prior to scheduled necropsy (i.e. Day 92 of the study). This procedure was discussed with the Belgian authorities and accepted (information provided by the Sponsor).
Details on study schedule:
- F1 parental animals not mated until 11-13 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age or shortly thereafter.
- Age at mating of the mated animals in the study: 15-16 weeks (P, F0), 14-16 weeks (F1)
Doses / concentrationsopen allclose all
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
actual ingested, dose volume: 5mL/kg body weight (actual dose volumes were calculated according to the latest body weight).
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
actual ingested, dose volume: 5mL/kg body weight (actual dose volumes were calculated according to the latest body weight).
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
actual ingested, dose volume: 5mL/kg body weight (actual dose volumes were calculated according to the latest body weight).
No. of animals per sex per dose:
F0-parental animals: 24 per sex per dose.
F1-parental animals: 24 per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected based on preliminary results of a dose range finding study (NOTOX Project 48923, which was part of the teratology rat study NOTOX Project 489241). In this study animals were treated at 0, 150, 450 or 1000 mg/kg body weight/day for a period of 14 days. Incidentally, piloerection, salivation and dark faeces were noted for animals at 450 and/or 1000 mg/kg. As compared to the control animals, lower food intake was recorded at all dose levels on several days during the first week of treatment. As a consequence, body weight loss was seen followed by partial recovery. Based on this information, dose levels of 0, 150, 450 and 1000 mg/kg were selected.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS:
Mortality / Viability: At least twice daily.
Clinical signs: At least once daily, detailed clinical observations were made in all animals. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.

BODY WEIGHT: 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, 4, 7, 14 and 21.

FOOD CONSUMPTION: Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1, 4, 7, 14 and 21.

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Estrous cyclicity (parental animals):
Estrous cycle determination: Daily vaginal lavage was performed to determine the stage of estrus beginning 21 days prior to initiation of the mating period and until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also performed to determine the stage of estrus. An analysis of the female cycle pattern was reported.

Reproduction processes: Male number paired with, mating date, confirmation of pregnancy, and delivery date were recorded.
Sperm parameters (parental animals):
For all surviving F0- and F1-males of Groups 1-4, the following assessments were performed:
Sperm samples were taken from the proximal part of the vas deferens (right):
- Sperm motility was assessed from all samples. In the case of F1-males, this was performed in a blinded manner.
- Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal were recorded. Evaluation was performed in a blinded manner.

One testis and one epididymis (left) from all males were removed, placed in labeled bags, and kept in the freezer at <-15°C. After thawing the left testis and epididymis were weighed, homogenized and evaluated for sperm numbers. Evaluation was performed in a blinded manner.
Litter observations:
STANDARDISATION OF LITTERS
To reduce variability among the litters, on Day 4 of lactation or shortly thereafter, eight pups from each litter of equal sex distribution (if possible) were randomly selected. For litters consisting of fewer than eight pups, adjustments for litter size were not performed.

PARAMETERS EXAMINED
The following parameters were examined in the offspring (F1- / F2-pups):
- Number and sex of pups/litter
- Mortality / Viability: The numbers of live and dead pups at the First Litter Check (= check at 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 in all animals.
- Body weights: Live pups were weighed during lactation on Days 1, 4, 7 and weekly thereafter.
- Sex: Was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).
- Balanopreputial separation: Each selected male pup (24 rats/group) was observed for balanopreputial separation (prepuce opening) beginning on postnatal Day 35 (Korenbrot, 1977). Examination of the males was continued daily until balanopreputial separation was present. The body weight of each male was recorded on the day of acquisition of balanopreputial separation.
- Vaginal perforation: Each selected female pup (24 rats/group) was observed for vaginal perforation (vaginal opening) beginning on postnatal Day 25 (Adams, 1985). Examination of the females was continued daily until vaginal perforation was present. The body weight of each female was recorded on the day of acquisition of vaginal perforation.
- Anogenital distance: Anogenital distance was measured for all live F2-pups on Day 1 of lactation.


GROSS EXAMINATION OF DEAD PUPS:
Stillborn pups and pups dying between birth and Day 4 of lactation were sexed and dissected (including the heart and the brain examined by a mid-coronal slice) by a technique described by Stuckhardt and Poppe (Stuckhardt, 1984).
For pups found dead or killed in extremis from Day 5 of lactation to weaning a gross necropsy was performed (including sex determination) and gross lesions were saved for possible future histopathological examination.
Postmortem examinations (parental animals):
Half of the F0-males were sacrificed two days before the first delivery of litters, the other half was sacrificed on the first day of deliveries. The reason to start necropsy approximately one week earlier than originally planned was a shortage of test substance. In consultation with the Sponsor it was decided to cancel dosing of the F0-males. In this way, sufficient test substance was left to continue dosing of the F0-females. The F0-males were sacrificed as soon as possible. The omission of one or two days of dosing before planned necropsy will not influence the study integrity.

F1-males were killed 2 to 20 days after delivery of the litters. The delay in necropsy by 1-3 weeks was caused by the fact that in the F1-generation, litter size was statistically significantly lower in the high dose group as compared to the control group. According to the OECD guideline for the two-generation study (OECD 416) it is recommended to consider to remate the F1-adults to produce a second litter in the case of a treatment-related alteration in litter size. Therefore, in consultation with the Sponsor necropsy of the F1-males was postponed. A thorough evaluation of the reproduction data obtained from the F0- and F1-generation so far was performed. Based on the data obtained from the F0-generation which also indicated a treatment-related effect on male reproduction, it was decided in consultation with the Sponsor that a second mating with the F1-males will not be of an additional value. Therefore, it was decided not to remate the F1-males, but to send them to necropsy.

F0-and F1-females were killed on Day 21 post partum or shortly thereafter.

Females showing no evidence of copulation were killed approximately 21 days after the last day of the mating period.
Non-pregnant females were killed on Day 25-27 post-coitum.

In case a female was not pregnant, the uterus was stained using the Salewski technique (Salewski, 1964) in order to determine any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).


GROSS NECROPSY
After sacrifice or death 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.

HISTOPATHOLOGY / ORGAN WEIGHTS F0 and F1 parental:
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Kidneys, Testes, Liver, Thyroid, Ovaries, Uterus, Pituitary.

The following tissues were examined microscopically from 10 animals/sex of control and high dose F0 and F1 groups and animals 192 (killed in extremis) and 266 (found dead): cervix, coagulation gland, epididymidis, ovaries, prostate gland, seminal vesicles, stomach, testis, uterus, vagina, all gross lesions. The following tissues were not examined microscopically since no signs of toxicity or target organ involvement were noted at macroscopic examination: adrenals, brain (cerebellum, mid-brain, cortex), kidney, liver, pituitary gland, spleen, thyroid including parathyroid (if detectable).

In additon, histopathology was performed on the reproductive organs of all F0 and F1 animals that failed to mate, sire or deliver healthy offspring
The testes and epididymides or uterus of 10 randomly selected animals of the intermediate dose groups of the F0- and F1-generation.
All gross lesions of all animals.
Postmortem examinations (offspring):
SACRIFICE
The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age or shortly thereafter.
These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
All nonselected F1- and F2-weanlings were sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs with emphasis on developmental morphology. Descriptions of all macroscopic abnormalities were recorded. All gross lesions were collected and placed in 10% buffered formalin.

GROSS NECROPSY
Gross necropsy consisted of external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs with emphasis on developmental morphology.

HISTOPATHOLOGY / ORGAN WEIGTHS
Brain, thymus and spleen were weighed and fixed in 10% buffered formalin for possible future analysis..
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (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 (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.
- The T-test (Gossett, 1908) was applied for sperm concentrations in the testis and epididymis when comparing Groups 1 and 4. Since a treatment-related effect was noted, it was decided to perform statistical evaluation on all groups. For this the Dunnett-test (see above) was used.
- The percentage of motile spermatozoa, progressive motile spermatozoa (F1-generation only), sperm with normal morphology, and number of implantation sites in the F0-generation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal, 1952) to determine intergroup difference. If the results of the ANOVA were significant (p<0.05), the Wilcoxon test (Wilcoxon, 1945) was applied to the data to compare the treated groups to the control group.
- For the statistical analysis of selected microscopic findings the Fisher Exact-test and Kruskal-Wallis nonparametric ANOVA test were used.

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 might have been rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each dose group reproduction parameters were expressed in two ways:
- As a mean (with standard deviation) of the number observed for each litter
- Relative to a second parameter and calculated on a total group basis

For each group the following calculations were performed:

Percentage mating males = (Number of males mated / Number of males paired) x 100
Percentage mating females = (Number of females mated / Number of females paired) x 100
Fertility index males = (Number of males generating a pregnancy / Number of males paired) x 100
Fertility index females = (Number of pregnant females / Number of females paired) x 100
Conception rate = (Number of pregnant females / Number of females mated) x 100
Gestation index = (Number of females bearing live pups / Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
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 post-partum = (Number of dead pups on Day 4 post partum / Number of live pups at First Litter Check) x 100
Percentage of breeding loss Day 5 until weaning
= (Number of dead pups between Days 5 and 21 post partum / Number of live pups on Day 4 post partum) x 100
Percentage live males at weaning = (Number of live male pups on Day 21 post partum / Number of live pups on Day 21 post partum) x 100
Percentage live females at weaning = (Number of live female pups on Day 21 post partum / Number of live pups on Day 21 post partum) x 100
Viability index = (Number of live pups on Day 4 post partum / Number of pups born alive) x 100
Weaning index = (Number of live pups on Day 21 post partum / Number of live pups on Day 4 post partum) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were noted which were considered toxicologically relevant.
From Day 67 of the pre-mating period until the last day of dosing, both lethargy and a fluffy fur at a very minor degree were noted for males treated at 1000 mg/kg (data not shown in the tables). The severity of both findings was too low as to record online, but high dose males could be clearly distinguished from control animals. Due to the very low severity and the absence of similar observations for males in the F1-generation, no toxicological relevance was attributed to these findings.
Slight to severe salivation was noted in one male and one female treated at 150 mg/kg and all males and females treated at 450 and 1000 mg/kg on one or more days during the study period. This finding was considered to be related to the taste of the formulation rather than a toxic effect.
Incidental findings consisted of pale appearance and/or piloerection in one female each in the control (no. 108) and the high dose group (no. 180), rales in one male at 450 mg/kg (no. 72), and hunched posture in one male at 1000 mg/kg (no. 75). Given the limited incidence of occurrence in singular animals, the transient nature of the findings and/or the fact that control animals were also affected, these clinical signs were considered to have occurred by chance and are not reflections of treatment-related toxicity.
Other incidental findings consisted of alopecia, scabbing, scales and/or wounds on several body parts, focal erythema of the left inguinal region, a dark eye, and watery discharge from the eye. These findings are occasionally noted in rats of this age and strain; as they were considered to be within the range of background findings among rats housed and treated under conditions of this study, these findings are not considered signs of toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
On the morning of Day 52 of treatment (28 January 2009), female no. 192 (Group 4) was found with a severe wound in the abdominal region. This female was housed together with female nos. 189-191. One or more of these animals must have attacked female no. 192. In the light of the severity of the wounds, it was decided to sacrifice this animal for humane reasons. Findings at necropsy included missing jejunum (cannibalism) and alopecia (left cheek). Based on the observations recorded for female no. 192 during the study, there was no reason to expect this incident: clinical signs comprised of alopecia and scabs of the left cheek, which were noted from Week 7 of treatment onwards. In addition, slight salivation was recorded from Week 4 of treatment onwards. Body weight and food consumption were unaffected by treatment. Therefore, no explanation can be given for this incident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased body weights and body weight gains were observed for males and females treated at 1000 mg/kg. In males, body weights were significantly decreased from Day 36 of the pre-mating period onwards including the mating and post-mating period. Body weight gain was significantly lower during the entire pre-mating period. In females, body weights were significantly lower on Day 64 of the pre-mating period, Day 1 of the mating period, Days 17-20 of the post-coitum period and during lactation. Body weight gain was significantly lower on Day 64 of the pre-mating period and Day 20 post-coitum.
No treatment-related changes in body weight and body weight gain were noted for males and females treated at 150 and 450 mg/kg.
The significantly lower body weight gain noted in males treated at 450 mg/kg on Day 8 of the pre-mating period is not considered to be toxicologically relevant, since this change was slight and transient in nature.
Females treated at 150 mg/kg showed a significantly higher body weight gain on Day 4 of the lactation period as compared to controls. This finding is considered to be of no toxicological significance since the opposite effect would have been expected in the case of toxicity.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A trend towards lower absolute food intake was seen in females at 1000 mg/kg during the post-coitum period. However, changes were very slight and not always statistically significant. Absolute food intake was significantly lower from Day 4 of lactation onwards. After allowance for body weight, food consumption was significantly lower from Day 7 of lactation onwards.
The slightly lower food intake noted in males and females at 1000 mg/kg on Days 1-8 and/or Days 57-64 during the pre-mating period was not considered toxicologically relevant since changes were very slight as compared to controls.
In females at 450 mg/kg absolute food consumption was slightly higher on Days 1-8 and Days 22-29 during the pre-mating phase. In addition, significantly higher relative food consumption was recorded incidentally for males and females at 450 mg/kg in the pre-mating period. This finding is considered to be of no toxicological relevance since the opposite effect would have been expected in case of toxicity.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Terminal body weight was statistically significantly lower in males at 1000 mg/kg as compared to control males. No treatment-related changes in terminal body weights were noted in males at 150 mg/kg and 450 mg/kg and females up to 1000 mg/kg.
No toxicologically significant changes were noted regarding organ weights and organ to body weight ratios.
The following statistically significant changes in organ weights distinguished treated from control animals:
- Decreased absolute brain weight in males at 450 mg/kg and 1000 mg/kg and increased brain to body weight ratio in males at 1000 mg/kg.
- Increased liver to body weight ratios in males at 1000 mg/kg and females at 150 and 1000 mg/kg (not statistically significant in females at 450 mg/kg).
- Increased kidney to body weight ratio in males at 450 and 1000 mg/kg.
- Increased absolute adrenal weight and adrenal to body weight ratio in females at 1000 mg/kg.
- Decreased absolute spleen weight and spleen to body weight ratio in females at 1000 mg/kg.
- Decreased absolute prostate weight in males at 1000 mg/kg.
- Decreased seminal vesicle weight in males at 150 mg/kg (before and after correction for terminal body weight) and 1000 mg/kg (absolute).
- Decreased absolute ovaries weight in females at 1000 mg/kg.
- Increased testes to body weight ratio in males at 1000 mg/kg.
These changes were slight, were in the normal range of biological variation noted for rats of this strain and age and/or were caused by the relatively low terminal body weight. In the absence of any corroborative microscopic findings and/or dose-response relationship, they were considered to be of no toxicological significance.
The relatively high uterus weights recorded for two females at 1000 mg/kg (nos. 177 and 179) corresponded with the necropsy findings of one late resorption and fluid content in the uterus, respectively. The high spleen weight recorded for control female no. 108 and low weights of testes and epididymides recorded for one male at 150 mg-kg (no. 27) was in agreement with the macroscopic observation of enlarged spleen and small testes and epididymides, respectively.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
At 450 mg/kg one female (no. 156) showed two early resorptions and another (no. 158) showed dark red fluid in the left uterus horn. For one female at 1000 mg/kg (no. 177) one late resorption was recorded. At the low incidence and in the absence of a treatment-related distribution, they were considered to be changes without toxicological significance.
Other incidental findings included a reddish focus in the stomach, thickened wall of the duodenum, enlarged liver (caudate lobe and papillary processes), and liver reduced in size (left median and lateral lobe), accessory liver and pale discoloration of the liver, black-brown or pale discoloration of the adrenal glands, a watery-clear cyst in the kidney, and enlarged spleen. Also noted were testes, epididymides and/or seminal vesicles reduced in size, alopecia, and bent tail apex. These macroscopic findings were considered to be spontaneous in nature and not to represent signs of toxicity.
Uterus containing fluid was noted for one female each in the control group and all treatment groups. This is a common finding related to a stage in the estrous cycle and does not reflect any sign of toxicity.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Morphologic alterations due to treatment were noted in the testis, epididymis, uterus and vagina in both generations. These consisted of:
- testis: multifocal seminiferous epithelial degeneration at minor degrees (minimal or slight) of severity were increased in Group 4 (1000 mg/kg/day) animals of both generations without statistical significance but with a positive trend p < 0.01.
- epididymis: seminiferous cell debris at a minimal or slight degree paralleled the incidence of seminiferous degeneration in the testes of Group 4 animals and also demonstrated a positive trend in both generations.
These findings were regarded as indicators of minimal testicular toxicity.
- uterus: attenuated myometrium at a minimal to moderate degree in the F0 and at minimal or slight degree in F1 was slightly increased in Group 4 with statistical significance only in F0 Group 4 p < 0.1 with a positive trend p < 0.01.
- vagina:
- epithelial mucification at a minimal to moderate degree was increased in all treated groups of the F0, however was statistically significant p < 0.05 only in Group 3 and there was no trend. In F1 this finding was increased in Group 4, statistically significant p < 0.1 with a positive trend p < 0.05.
- attenuated epithelium at slight or moderate degree was increased only in F0 Group 4 rats, p < 0.05 and there was a positive trend p < 0.01.
The finding of attenuated myometrium in the uterus was considered to be a secondary effect due to reduced body weight gain and the findings in the vagina may have reflected a slower return to normal estrus cycles in the treated females.
In animals suspected of infertility there were no treatment related findings which were considered to be responsible for the lack of reproductive performance.
Quantitative assessment of primordial follicles in the ovaries of F1 females revealed no difference between Group 1 and Group 4.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Breeding parameters:
Breeding parameters were unaffected by treatment up to 1000 mg/kg. No deficiencies in maternal care were observed.
The significantly lower percentage of male pups as compared to female pups recorded in the mid dose group (450 mg/kg) at first litter check was not considered toxicologically-relevant as no dose-response relationship was observed.

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of females per group that were classified as having a regular cycle was 100.0, 87.5, 100.0, and 82.6% at 0, 150, 450 and 1000 mg/kg, respectively. Two females each at 150 mg/kg (nos. 126, 127) and 1000 mg/kg (nos. 185, 189) showed an irregular cycle. Extended estrus was recorded for one female each at 150 mg/kg (no. 138) and 1000 mg/kg (no. 171). Furthermore, an acyclic cycle was noted in one female in the high dose group (no. 181).
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Following treatment with the test substance, a statistically significantly decrease in left epididymis organ weight (1000 mg/kg), sperm concentration of the left epididymis (450 and 1000 mg/kg) and left testis (450 mg/kg) were noted. Moreover, the quality of sperm was affected by treatment at 450 mg/kg and 1000 mg/kg. In both groups, motility score and percentage of sperm cells with normal morphology were statistically significantly lower as compared to controls. The deterioration of sperm morphology was comparable for the mid and high dose and consisted of a marked increase in the number of sperms with separated head.
At 150 mg/kg (Group 2), no treatment-related changes were noted for organ weight of epididymis (left) and testis (left), sperm concentration or sperm morphology. At the individual level, there was a trend towards lower sperm motility scores resulting in a very slight, but statistically significantly lower median value for Group 2 as compared to the control group. However, this change consisted mainly of a shift from very good towards good motility. Moreover, the motility scores for control group animals in this study were at the upper limit of the range normally seen for animals of this age and strain used under comparable conditions. Therefore, sperm motility in the low dose group was considered to be within the normal range of biological variation.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Reproduction parameters were unaffected by treatment.
No signs of difficult or prolonged parturition or signs of abortion or premature birth were noted among the pregnant females.
All fertility parameters, including the number of implantation sites, were well within the normal range of variation in rats of this age and strain. The statistically significantly higher number of implantation sites noted at 150 mg/kg (Group 2) as compared to controls was considered to be a fortuitous finding. In case of toxicity a decrease rather than an increase in the number of implantation sites would have been expected.
In litter no. 117 all pups were dead at first litter check. Therefore, for this female body weight and food consumption during lactation were excluded from the summary tables. Furthermore, in litter no. 110, 14 out of 15 pups were either missing or dead at first litter check. Since both litters were in the control group, these findings were not related to treatment with XTJ-568 dihydrochloride.A significant decrease in organ weight (left epididymis and testis) and sperm concentration (left epididymis) was noted in males treated at 1000 mg/kg. Moreover, sperm quality was affected by treatment as reflected by decreased motility and progressive motility at 1000 mg/kg and a lower percentage of sperm cells with normal morphology at 450 and 1000 mg/kg. The latter change in normal morphology was comparable for the mid and high dose group and was caused by a marked increase in the number of sperms with separated head.
In the low dose group treated at 150 mg/kg sperm parameters were comparable with those of the control group.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
450 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: Based on parental toxicity at 1000 mg/kg bw/d.
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
450 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
reproductive function (sperm measures)
reproductive performance
other: Based on parental toxicity and various reproductive findings at 1000 mg/kg bw/d. Refer to 'conclusions' section for more information.
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
450 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
other: Based on reduced body weights of F0 and F1 pups, and reductions in F1 litter size and mean number of living F1 pups, at 1000 mg/kg bw/d.

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: male and female reproductive system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were noted which were considered toxicologically-relevant.
Slight to moderate salivation was noted in 0/24, 1/24, 21/24 and 24/24 males and 0/24, 0/24, 15/23 and 22/23 females of Groups 1, 2, 3 and 4, respectively, on one or more days during the study period. This finding was considered to be related to the taste of the formulation rather than a toxic effect.
Piloerection and/or hunched posture were noted in one female in the control group (no. 313) and two females in the high dose group (nos. 387 and 392) on one or a few days during treatment. Given the limited incidence of occurrence in singular animals, the transient nature of the findings and/or the fact that also control animals were affected, these clinical signs were considered to be spontaneous in origin.
For male no. 245 (Group 2) a wound in the neck was noted on 04 June 2009 (pre-mating period). This finding was preceded by scabbing. A health inspection was performed by a veterinary on 05 and 10 June 2009, and the wound was disinfected daily (except for 22 June 2009) with 10% Betadine solution (VIATRIS Manufacturing B.V., Diemen, The Netherlands). On 24 June 2009 (pre-mating period), the animal had recovered. There was no reason to suspect a treatment-related effect.
Other incidental findings consisted of alopecia, scabbing, scales and/or wounds on several body parts, chromodacryorrhoea of the eye and/or periorbital region, and broken tail apex. As these findings are occasionally noted in rats of this age and strain housed and treated under conditions of this study, they were not considered signs of toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two female pups of Groups 3 and 4 (nos. 345 and 374, respectively) selected for the F1-generation died spontaneously (for details see mortality offspring.). This was before the start of the second generation. Due to computer technical reasons the date of death is listed as 20 April 2009 in the mortality tables.
One male (no. 266) treated at 450 mg/kg was found dead on Day 49 of the pre-mating period. No clinical signs were noted before death. At necropsy, beginning autolysis and many dark-red foci on the thymus were noted. Due to the isolated nature of this death and in the absence of any dose-response, it was considered to be unrelated to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated at 450 mg/kg (Group 3) and 1000 mg/kg (Group 4) showed lower body weights as compared to controls during the whole observation period. Significantly lower values were noted for Group 3 animals from Days 29-57 pre-mating and for Group 4 animals during the entire pre-mating, mating and post-mating periods. This effect was due to a developmental delay which had occurred during the lactation period. After correction for the relatively low body weights recorded for Groups 3 and 4 males on Day 1 of the pre-mating period, body weight gain was comparable between the treated and control groups. The significantly lower body weight gain noted in males treated at 1000 mg/kg on Day 8 of post-mating was considered to be of no toxicological significance, as this change was relatively small and transient.
Females treated at 1000 mg/kg (Group 4) showed a trend towards lower body weights as compared to controls; reductions in body weight reached statistical significance during Days 1-8 of pre-mating and from Day 20 post-coitum until Day 7 of lactation. Body weight gains were significantly lower on Days 17-20 post-coitum only.
The higher body weight gain observed for females treated at 450 mg/kg during the whole pre-mating period was at least in part attributable to the comparatively low initial weights of female nos. 346 and 349. Over the pre-mating period both females gained enough weight to (mostly) normalize with the other females in this group. As such, the amount of weights gained by these two females resulted in comparatively high body weight gain percentages thereby driving the overall group mean body weight gains to statistical significance. In addition, higher body weight gains were recorded for females at 1000 mg/kg during pre-mating (not statistically significant) and females at 450 mg/kg on Day 14 of post-coitum. These changes were considered to be no sign of toxicity since if any adverse effects were present the opposite effect would have been expected.
No toxicologically significant changes in body weights and body weight gain were noted at 150 mg/kg.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, absolute food consumption was significantly lower for males than controls throughout the entire pre-mating period and on Days 1-29 post-mating. This effect was a direct cause of the initial lower body weight of these high dose animals as compared to controls. After correcting for body weight, relative food consumption was even higher at 1000 mg/kg than in controls during the whole observation period (not always statistically significant). In males treated at 450 mg/kg, a similar trend was noted for relative food consumption.
For females treated at 1000 mg/kg, a trend towards higher relative food consumption as compared to controls was noted during the pre-mating period (not always statistically significant) and on Days 11-14 post-coitum. Significantly lower food intake (before and after allowance for body weight) was recorded during the lactation period. Females treated at 450 mg/kg showed a higher food intake (before and after allowance for body weight) on Days 11-14 post-coitum. The latter finding is considered to be of no toxicological relevance since the opposite effect would have been expected in the case of toxicity.
Food consumption before or after allowance for body weight was similar between animals (both sexes) treated at 150 mg/kg and control animals.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Terminal body weight was statistically significantly lower in males at 1000 mg/kg as compared to control males. No treatment-related changes in terminal body weights were noted in males at 150 mg/kg and 450 mg/kg and females up to 1000 mg/kg.
No toxicologically significant changes were noted regarding organ weights and organ to body weight ratios.
The following statistically significant changes in organ weights distinguished treated from control animals:
- Decreased absolute brain weight in males and females at 1000 mg/kg and increased brain to body weight ratio in males at 1000 mg/kg
- Slightly increased pituitary gland to body weight ratios in males at 1000 mg/kg.
- Decreased absolute liver weights in males at 1000 mg/kg, increased absolute liver weights in females at 450 mg/kg, increased liver to body weight ratios in males at 450 and 1000 mg/kg and females at 450 mg/kg.
- Slightly decreased thyroid to body weight ratio in females at 450 mg/kg.
- Decreased absolute kidney weights and increased kidney to body weight ratio in males at 1000 mg/kg.
- Increased adrenal to body weight ratios in males at 1000 mg/kg.
- Decreased absolute spleen weight in males at 1000 mg/kg.
- Decreased absolute testes weight and increased testes to body weight ratios in males at 1000 mg/kg.
- Decreased absolute prostate weight in males at 1000 mg/kg.
- Decreased absolute epididymides weight in males at 450 and 1000 mg/kg.
- Decreased absolute seminal vesicle weight in males at 150 mg/kg (before and after allowance for terminal body weight) and at 1000 mg/kg (before allowance for terminal body weight).
These changes were slight, were in the normal range of biological variation noted for rats of this strain and age and/or were caused by the relatively low terminal body weight of high dose males. In the absence of any corroborative microscopic findings and/or dose-response relationship, they were considered to be of no toxicological significance.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
For 3/24 males in the high dose group (1000 mg/kg) reduced size of testes (nos. 285, 288), epididymides (no. 285) and/or seminal vesicles (nos. 286, 288) was recorded. Correlating microscopic findings were oligospermia epididymis (no. 285), multifocal seminiferous epithelial degeneration testis (no. 285), hypotrophic acini seminal vesicle (no. 286). There were no corroborative microscopic changes for the small testis of male no. 288.
Incidental findings seen among control or treated animals included enlarged spleen, bent tail apex, black-brown and hard nodule in the liver, black-brown foci on the thymus, and alopecia of various body parts. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered to be changes of no toxicological significance.
Uterus contains fluid was noted for 2/24, 1/24, 2/23 and 0/23 females treated at 0, 150, 450 and 1000 mg/kg. This finding is a normal finding that correlates with a particular stage of the estrous cycle, and does not indicate any signs of treatment-related toxicity.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Morphologic alterations due to treatment were noted in the testis, epididymis, uterus and vagina in both generations. These consisted of:

testis:
- multifocal seminiferous epithelial degeneration at minor degrees (minimal or slight) of severity were increased in Group 4 (1000 mg/kg/day) animals of both generations without statistical significance but with a positive trend p < 0.01.

epididymis:
- seminiferous cell debris at a minimal or slight degree paralleled the incidence of seminiferous degeneration in the testes of Group 4 animals and also demonstrated a positive trend in both generations.

These findings were regarded as indicators of minimal testicular toxicity.

uterus:
- attenuated myometrium at a minimal to moderate degree in the F0 and at minimal or slight degree in F1 was slightly increased in Group 4 with statistical significance only in F0 Group 4 p < 0.1 with a positive trend p < 0.01.

vagina:
- epithelial mucification at a minimal to moderate degree was increased in all treated groups of the F0, however was statistically significant p < 0.05 only in Group 3 and there was no trend. In F1 this finding was increased in Group 4, statistically significant p < 0.1 with a positive trend p < 0.05.
- attenuated epithelium at slight or moderate degree was increased only in F0 Group 4 rats, p < 0.05 and there was a positive trend p < 0.01.

The finding of attenuated myometrium in the uterus was considered to be a secondary effect due to reduced body weight gain and the findings in the vagina may have reflected a slower return to normal estrus cycles in the treated females.

In animals suspected of infertility there were no treatment related findings which were considered to be responsible for the lack of reproductive performance.

Quantitative assessment of primordial follicles in the ovaries of F1 females revealed no difference between Group 1 and Group 4.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
No deficiencies in maternal care were observed.
Following treatment at 1000 mg/kg, litter size was significantly lower than controls. The mean number of living pups at first litter check was 7.9 in Group 4 as compared to 11.1 in Group 1. After culling, the mean number of living pups was 6.8 and 7.6 for Groups 4 and 1, respectively, on Days 4 and 21 post-partum.
Duration of gestation, dead pups at first litter check, postnatal loss and breeding loss, viability index and weaning index were not affected by treatment up to 1000 mg/kg.
Breeding parameters were unaffected by treatment at 150 and 450 mg/kg.

Reproductive function / performance (P1)

Reproductive function: estrous cycle:
effects observed, treatment-related
Description (incidence and severity):
The percentage of females per group that were classified as having a ¿regular¿ estrous cycle was 95.8, 100.0, 87.0 and 65.2% at 0, 150, 450 and 1000 mg/kg, respectively. In treatment Groups 1-4, the number of females with irregular estrous cycle was 1 (no. 310), 0, 2 (nos. 360, 366) and 5 (nos. 369, 377, 379, 385, 387), respectively. Extended di-estrus during pairing was noted for one female at 150 mg/kg (no. 323) and two females at 450 mg/kg (nos. 350, 354). Furthermore, one female at 450 mg/kg (no. 347) and three females in the high dose group (nos. 380-382) showed an acyclic cycle.

Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in organ weight (left epididymis and testis) and sperm concentration (left epididymis) was noted in males treated at 1000 mg/kg. Moreover, sperm quality was affected by treatment as reflected by decreased motility and progressive motility at 1000 mg/kg and a lower percentage of sperm cells with normal morphology at 450 and 1000 mg/kg. The latter change in normal morphology was comparable for the mid and high dose group and was caused by a marked increase in the number of sperms with separated head.
In the low dose group treated at 150 mg/kg sperm parameters were comparable with those of the control group.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
For one female (no. 391) in the high dose group, red discolored vaginal secretion was noted on Days 14 and 15 post-coitum (not shown in tables). Salewski staining of the uterus at necropsy showed no implantation sites. Therefore an abortion can be excluded for this female. The reason for the observed red discolored vaginal secretion is not known. However, due to the isolated character of this finding, it was considered to be no sign of toxicity.
No signs of difficult or prolonged parturition or signs of abortion or premature birth were noted among the pregnant females.
The number of implantation sites was significantly lower in the high dose group as compared to controls.

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction toxicity
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
reproductive function (estrous cycle)
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P1)

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: male and female reproductive system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted.
Incidental clinical symptoms consisted of small size, pale and cold appearance, no milk in the stomach, alopecia, scabbing and/or wounds of several body parts, chromodacryorrhoea or ptosis. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
In the control, low dose, mid dose and high dose group, the number of pups per litter which were found dead or missing during the first days of lactation was 24/5, 6/4, 5/5 and 7/5, respectively. In absence of a dose-response relationship, these deaths were not considered to be related to treatment.
Moreover, two pups selected for the F1-generation were found dead in the period between weaning and start of the F1-generation:
- Female pup no. 6 from litter no. 145 (Group 3) was found dead on Day 22 post-partum. No clinical signs were noted in the period before the spontaneous death. Macroscopic examination revealed perforation of the esophagus, indicating an oral gavage error being the cause of death. Moreover, autolysis was noted for this pup.
- Female pup no. 4 from litter 173 (Group 4) was found dead on Day 30 post-partum. No clinical signs were noted in the period before the spontaneous death. Macroscopic examination revealed cannibalism and autolysis.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group (1000 mg/kg), significantly lower body weights were noted for male and female pups from lactation Days 7 and 14, respectively, onwards. At birth, body weights were in the same range as those of the control animals.
Body weights of F1-offspring treated at 150 and 450 mg/kg remained in the same range as controls over the study period.
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:
effects observed, non-treatment-related
Description (incidence and severity):
The day of balanopreputial separation (BPS) and vaginal opening (VO) was delayed in pups of the high dose group (BPS: Day 44.6 at 1000 mg/kg compared to Day 42.5 in the control group; VO: Day 34.7 at 1000 mg/kg compared to Day 33.8 in the control group). Based on the fact that the mean body weights on the day of measurement for this group were lower than controls, these delays were considered secondary to the reduced body weights.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significantly lower spleen weights (absolute and relative to body weight) were recorded in pups of the high dose group (both sexes) as compared to controls. The biological significance of this finding in F1-offspring (both sexes) was doubted as there were no similar findings for F2-offspring.
The decreased absolute weights of thymus and brain and increased brain/body weight ratio noted at 1000 mg/kg (both sexes) and decreased weight of thymus (absolute) noted at 450 mg/kg (males) were considered to be secondary to the reduced terminal body weights.
Organ weights and organ to body weight ratios of F1-pups at 150 and 450 mg/kg were in the same range as those of control animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related macroscopic findings noted with exposure to the test substance up to 1000 mg/kg.
For pup no. 8 of litter no. 100 (Group 1) ureter dilation and pale discoloration of the right kidney was noted. Since it was a pup of the control group, this finding was considered to have occurred spontaneously.
Incidental findings included no milk in the stomach and small size. Additionally, cannibalism or autolysis was noted for dead pups. No relationship with treatment was established for these observations.
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduction in litter size and mean number of living pups (F1 generation) and reduced body weight of pups (F0- and F1-generation)

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically-relevant clinical signs noted for any treatment group.
Incidental findings seen among control and treated animals included small size, pale or cold appearance, no milk in stomach, missing tail apex, and alopecia of various body parts. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No treatment-related mortality occurred.
In the control, low dose, mid dose and high dose groups, the number of pups (litters) which were found dead or missing during the first days of lactation was 3(3), 2(2), 6(3) and 3(3), respectively. At the low incidence observed in the treatment groups and in absence of a dose response relationship, these deaths were not considered to be treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Although mean body weights were comparable for all groups (both sexes) on Day 1 of lactation, mean body weights of both males and females at 1000 mg/kg were significantly lower compared to controls on Days 14 and 21 post-partum.
No treatment-related changes were noted at 150 and 450 mg/kg.
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
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant changes were noted in organ weights and organ to body weight ratios.
The increased brain to body weight ratio noted for female pups at 1000 mg/kg was considered to be related to the lower body weights recorded for animals of the high dose group as compared to the control group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically-relevant macroscopic findings.
Incidental findings seen among control and treated animals included small size, no milk in the stomach, alopecia of different parts of the body, cannibalism and/or autolysis. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital distance:
Treatment up to 1000 mg/kg did not affect the anogenital distance of the F2-offspring.
The significantly lower median anogenital distance noted at 150 mg/kg (both sexes) and 450 mg/kg (males only) was not considered to be a sign of toxicity, as changes were very slight and no dose-response relationship could be established.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on reductions in litter size and mean number of living pups and reduced body weight of pups

Target system / organ toxicity (F2)

Critical effects observed:
no

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 in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Treatment with the substance by oral gavage in male and female Wistar Han rats at dose levels of 150, 450 and 1000 mg/kg revealed parental toxicity at 1000 mg/kg/day based on decreased body weights and/or body weight gains in females (both generations) and males (F1-generation), lower food consumption in females (both generations), reduced size of testes, epididymides and/or seminal vesicles in males (F1-generation), lower terminal body weights in males of both generations and morphological alterations recorded in testes and epididymides (both generations), uterus (F0-generation) and vagina (both generations). Based on these findings, the parental NOAEL was established as being 450 mg/kg/day.
Developmental toxicity was observed at 1000 mg/kg/day based on reductions in both litter size and mean number of living pups (F1-generation) and reduced body weight of pups (F0- and F1-generation). The definitive development NOAEL was established as being 450 mg/kg/day.
Reproduction toxicity was noted from 450 mg/kg onwards based on effects on sperm parameters in males (both generations: 450 and 1000 mg/kg), effects on estrous cycle (F0-generation: 1000 mg/kg; F1-generation: 450 and 1000 mg/kg) and decreased numbers of implantation sites in females (F1-generation: 1000 mg/kg). The reproduction NOAEL was established as being 150 mg/kg/day in the study report. However, the evaluations for reproductive toxicity were not conducted in a blinded fashion, and different protocols and equipment were used for the sperm motility evaluations. Most of the findings of abnormal sperm morphology were due to a separation of sperm heads from the remaining body of the sperm. These findings may have been an artifact of the sperm morphology preparation. From a practical perspective, the observed reduction in sperm numbers and quality did not affect the reproductive performance of the study animals at any dose level. On the basis of the parental toxicity observed at 1000 mg/kg and various reproductive findings at the 1000 mg/kg dose level, the reproductive toxicity NOAEL is considered to be 450 mg/kg. Given that the reproductive effects were observed at parentally toxic dosages, we conclude that XTJ-568 should not be considered to be a reproductive toxicant.