Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A two-generation study with the substance in rat was performed according to OECD Guideline 416, EPA OPPTS 870.3800 guideline and EU Method B.35 (Peter, 2010). Based on the findings, the definitive parental No Observed Adverse Effect Level (NOAEL) was established as being 450 mg/kg/day; the definitive reproduction NOAEL was established as being 450 mg/kg/day (instead of 150 mg/kg bw/d as concluded in the study report); the definitive development NOAEL was established as being 450 mg/kg/day.

Link to relevant study records
Reference
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
Qualifier:
according to guideline
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
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
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
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
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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
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)
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
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.
Oestrous 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
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
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: oestrous 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.
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.
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
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: oestrous 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.
Key result
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 (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Key result
Critical effects observed:
no
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
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)
Key result
Critical effects observed:
no
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.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
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
Key result
Critical effects observed:
no
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
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.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
chronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Two-generation toxicity study (OECD guideline 416)

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 the substance should not be considered to be a reproductive toxicant.

However, in the RAC Opinion proposing harmonised classification and labelling at EU level of the test substance, adopted 9 December 2016 (CLH-O-0000001412-86-132/F), is was considered that treatment with XTJ-568 dihydrochloride in this 2-generation toxicity study in rats resulted in a statistically significant decrease in the left epididymis weight (at the high dose, 1000 mg/kg bw/d). Microscopic examination showed, in the male high dose group, statistically significant minimal or slight multifocal seminiferous degeneration in the testis and minimal or slight seminiferous cell debris in the epididymis. The sperm evaluation confirmed these results and revealed a statistically significant decrease at 1000 mg/kg bw/d of sperm concentration in the left epididymis and left testis. The percentage of sperm cells with normal morphology was decreased and the number of sperm with separated head was increased (also observed at 450 mg/kg bw/d). A slightly lower sperm mobility was observed already at 150 mg/kg bw/d. The lack of effects on the testis in the repeated dose 13-week toxicity study at doses up to 150 mg/kg bw/d were not considered to negate those findings. Therefore classification for effects on fertility in category 2 was proposed (Repr. 2, H361f).

90 -day repeated dose toxicity study

The results of the subchronic 90-d oral toxicity study (Chase 2011) indicate that there are no effects on sperm number, motility or morphology at 150 mg/kg/day and support the conclusion that the substance is not a reproductive toxicant.


Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study with the substance in rat was performed according to OECD Guideline 414, EPA OPPTS 870.3700 guideline and EU Method B.31 (Peter, 2010). Based on the findings, the maternal No Observed Adverse Effect Level (NOAEL) for the substance was established as being 450 mg/kg/day. In the absence of treatment-related effects on reproductive parameters, fetal body weight and fetal morphology, a dosage level of 1000 mg/kg/day was considered to be the NOAEL for developmental toxicity.

A prenatal developmental toxicity study with the substance in rabbit was perforemd according to OECD Guideline 414 (Barnett, 2020). Based on the findings, the maternal NOAEL is considered to be 75 mg/kg/day while the developmental NOAEL is considered to be 100 mg/kg/day.

A two-generation study with the substance in rat was performed according to OECD Guideline 416, EPA OPPTS 870.3800 guideline and EU Method B.35 (Peter, 2010). Based on the findings, the definitive development NOAEL was established as being 450 mg/kg/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-01-05 - 2009-02-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Identification: XTJ-568 dihydrochloride
- Molecular formule: C10H26Cl2N2O2 (component A) C14H34Cl2N2O3 (component B)
- Molecular weight: 277.23 (component A) 349.34 (component B)
- Description: Clear yellow liquid (determined at NOTOX)
- Batch: 8666-080-C
- Purity: ~ 100%
- Composition: This product consists 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) is approximately 80% of the mixture and the 3/1 adduct (component B) is approximately 20%. The concentration of the salt in this solution is 70.5%.
- Test substance storage: At room temperature in the dark
- Stability under storage conditions: Stable
- Expiry date: 15 June 2009
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Rat Crl:WI(Han) (outbred, SPF-Quality) from Charles River, Sulzfeld, Germany
- Age at study initiation: Age at start of pairing: approximately 12 weeks. Females were nulliparous and non-pregnant at initiation of the study. Stock male rats Crl:WI(Han) (outbred, SPF-Quality) were used for mating with the females. After mating these males were placed back in their stock and might be used in further studies.
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing:
* Pre-mating: During acclimatization, females were housed in groups of 5 animals/cage in Macrolon cages (MIV type, height 18 cm). Acclimatization period was at least 5 days prior to pairing under laboratory conditions.
* Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm). During the weekend, mating procedures were suspended and the animals were housed in groups of maximum 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
* Post-coitum: Females were individually housed in Macrolon cages (MIII type, height 18 cm).
* Randomization: Upon detection of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups. Females which were mated on the same day were classified in the same subgroup.
* General: Sterilized 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 (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.
- Water (e.g. ad libitum): Free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: No data

Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3ºC (actual range: 18.6-22.8ºC)
- Humidity (%): 30-70% (actual range: 22-66%)
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
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.
Stability of test substance in vehicle: At least 8 days, if stored in the refrigerator (determined as part of NOTOX Project 489273).
Method of formulation: Formulations (w/w) were prepared weekly and were homogenised to a visually acceptable level. Adjustment was made for density of the test substance (1.106 g/mL).
Storage conditions: In the refrigerator.

Dose volume:
5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Concentrations of formulations:
0, 30, 90 and 200 mg/mL.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were done on one day during the treatment period according to a validated method (NOTOX Project 489272) as specified below.

Group Analysis (type of sample)

1 acc (M)
2 acc + hom (TMB)
3 acc (M)
4 acc + hom (TMB)

Duplicate samples were analyzed
acc=accuracy, hom=homogeneity, T=top, M=middle, B=bottom position of container

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was 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 90% and 110%).

A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation. The maximum contribution based on area to the Group 2 samples was 1.2%. The source for the response in Group 1 is probably due to carry-over of the analytical method, as no analytical column is used. Due to the nature of the test substance, this could not be circumvented.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation 10%).
Details on mating procedure:
Age at start of pairing: approximately 12 weeks
Breeding procedures:
One female was placed on a one-to-one-basis in the cage of a male rat. 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 female was separated from the male. When sufficient mated females had been obtained for each dose group, the surplus females were removed from the study.
Duration of treatment / exposure:
From Day 6 to Day 19 post-coitum, inclusive
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.
Duration of test:
20 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
450 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 females/sex/dose
Control animals:
yes, concurrent vehicle
Maternal examinations:
Mortality / Viability
At least twice daily.

Clinical signs
At least once daily from Day 0 post-coitum onwards. The time of onset, degree and duration was recorded. All symptoms were graded according to fixed scales: Maximum grade 1: grade 0 = absent, grade 1 = present.
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females.

Body weights
Days 0, 3 and 6-20 (daily) post-coitum.

Food consumption
Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

Water consumption
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
Ovaries and uterine content:
All animals were subjected to an examination post-mortem. All animals surviving to Day 20 post-coitum were sacrificed using an oxygen/carbon dioxide procedure. External, thoracic and abdominal examinations were performed for the detection of macroscopic abnormalities. All abnormalities were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the gravid uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy)
and also from gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.

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)).

The female genital tract including the placentas was preserved in 10% buffered formalin for possible histopathological examination.

Fetal examinations:
External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.

External:
Each viable fetus was examined in detail, sexed and weighed. All live fetuses were euthanized by subcutaneous injection of 0.01 mL pentobarbital (Euthesate®; Ceva Sante Animale B.V., Maassluis, The Netherlands for necropsies before 01 February 2009; Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands for necropsies from 01 February 2009 onwards) in the area between the scapulas. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined, crown-rump length measured, weighed and sexed. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed and the tissue was discarded.

Subsequently, each litter was divided into two groups:
- Approximately one half of the fetuses in each litter was examined for visceral anomalies (for details see below).
- The remaining half of the fetuses in each litter was used for skeletal examination (for details see below).

Visceral (Internal):
Approximately one-half of the fetuses in each litter was examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (Stuckhardt, 1984). This examination included the heart and major vessels. The sex of all fetuses was confirmed by internal examination.

The heads of these fetuses were removed and placed in Bouin's solution (Klinipath, Duiven, The Netherlands) for subsequent processing and soft-tissue examination using the Wilson sectioning technique (Wilson, 1965). After examination, the tissues were stored in 10% buffered formaline.

The carcasses were eviscerated and stored in identified containers containing 96% aqueous alcohol (Klinipath, Duiven, The Netherlands) without further examinations.

Skeletal:
Following confirmation of the sex by internal examination, the carcasses of the remaining half of the fetuses in each litter were eviscerated and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.

Following fixation in alcohol, all fetuses were macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson (Dawson, 1926). For the selected fetuses, the skeletal examination was performed. The specimens were archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Merck, Darmstadt, Germany) as preservative.
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 (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.

Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral, skeletal and combined), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis 1952) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn¿s test (Dunn 1964) was used to compare the compound-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.
The following definitions were applicable for implantation data:
- Fetal (late) resorptions were defined as a dead fetus with external degenerative changes and presence of distinguishable features such as head or limbs.
- Embryonic (early) resorptions were defined as evidence of implantation without presence of distinguishable features such as head or limbs.
- Post-implantation loss included embryonic (early) resorptions, fetal (late) resorptions and dead fetuses.

Historical control data:
Yes, available
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, slight body weight loss occurred during the first two days of treatment. Lower body weights and body weight gain as compared to controls were noted from the second day of treatment onwards. In addition, (for uterus weight) corrected body weight gain was statistically significantly decreased for the high dose group as compared to control animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, a lower food intake (before and after allowance for body weight) as compared to the control group was recorded during the whole treatment period (not always statistically significant). Effects were most pronounced in the first week of treatment.
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:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
450 mg/kg bw/day
Basis for effect level:
body weight and weight gain
other: based on reduced body weights and body weight gain of the high dose animals at 1000 mg/kg/day
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
> 1 000 mg/kg bw/day
Basis for effect level:
other: In the absence of treatment-related effects on reproductive parameters, fetal body weight and fetal morphology, a dosage level of 1000 mg/kg/day was considered to be the NOAEL for developmental toxicity.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Remarks:
embryotoxicity
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on the results in this prenatal developmental toxicity study, the maternal No Observed Adverse Effect Level (NOAEL) for the test substance was established as being 450 mg/kg/day, based on reduced body weights and body weight gain of the high dose animals at 1000 mg/kg/day. In the absence of treatment-related effects on reproductive parameters, fetal body weight and fetal morphology, a dosage level of 1000 mg/kg/day was considered to be the NOAEL for developmental toxicity.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2019-10-23 to 2020-04-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: T105 (log number 12189)
- Chemical name: Reaction mass of 1-[2-(2-aminobutoxy)ethoxy]but-2-ylamine and 1-({[2-(2-aminobutoxy)ethoxy]methyl}propoxy) but-2-ylamine; monoethyleneglycol, polybutyleneoxide-diamine (MEGPBOD)
- CAS number: 897393-42-9
- EC number: 447-920-2
- Appearance: liquid
- Expiration date of the lot/batch: 31 July 2021
- Purity: 98.5%
- Purity test date: July 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept at controlled room temperature, blanketed under nitrogen
- Stability under storage conditions: No data
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: the test substance is stable in the control substance when prepared and stored under the same conditions at concentrations bracketing those used in the present study (stability data documented in study number 1318012)
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The dosing formulations were prepared at least once weekly, blanketed with nitrogen and stored in a refrigerator set to maintain 4°C, and dispensed daily.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. facility located in Saint Constant, Canada
- Age at study initiation: 4 to 6 months
- Weight at study initiation: 3232 g to 4536 g at randomization and study assighment
- Fasting period before study: no
- Housing: The rabbits were individually housed in units of six to eight cages. No nesting materials were supplied because female rabbits were euthanized before parturition was expected.
- Animal enrichment: on the day of arrival, each rabbit was provided irradiated hay in the food hopper. Suspended chain link devices hanging from the stainless steel cages were supplied to all rabbits during the course of the study. Analyses for possible contamination were conducted on each lot of enrichment devices. It was considered that there were no known contaminants in the animal enrichment that would interfere with the objectives of the study.
- Diet (e.g. ad libitum): 150 g to 155 g (from the day of arrival), increased to 180 g to 185 g (from the first day of testing) of Certified Rabbit Chow®
- Water (e.g. ad libitum): Hydrogel™ water pack ad libitum supplemented by Hydrogel™ water pack for the first 7 days after arrival, and used at the discretion of the Staff Veterinarian and/or Study Director after these first 7 days
- Acclimation period: the rabbits were acclimated prior to initiation of dose administration (period is not specified)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68°F to 79°F (20°C to 26°C)
- Humidity (%): 30% to 70%
- Air changes (per hr): minimum of 10 changes per hour of fresh air that had been passed through 99.97% HEPA filters.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 2019-10-25 To: 2019-11-22
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared at least once weekly, blanketed with nitrogen and stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulation were also stirred continuously during dosing.

VEHICLE
- Concentration in vehicle: 0, 5, 10, 15, 20 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): RO deionized water is available from a continuous source at the Testing Facility
- Purity: not specified
- Preparation of the control substance: The control substance was prepare once weekly, stored in a refrigerator set to maintain 4 °C, and dispensed daily. The prepared control substance was removed from the refrigerator and stirred for at least 30 minutes before dosing. The control substance was also stirred continuously during dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sample collection and analysis:
Dose formulation samples were collected for analysis as follows:
• first preparation:
concentration: all groups
homogeneity: groups 2 and 5
stability: not applicable
• last preparation:
concentration: all groups
homogeneity: not applicable
stability: not applicable
All samples to be analyzed were shipped (on cold packs) to CRL, Ashland, OH for analysis.
Analyses were performed by GC using a validated analytical procedure (1318.DIW1.01)
Concentration and Homogeneity Analysis: Duplicate (1 mL) sets of top, middle, and bottom test substance samples (duplicate middle only for groups 1, 3 and 4) for the sampling time points were shipped to the analytical laboratory; triplicate sets of top, middle and bottom test sample analysis (triplicate middle only for groups 1, 3 and 4) were collected in the same manner and stored in a refrigerator set to maintain 4 °C, blanketed under nitrogen, at the Testing Facility, as backup samples until utilized for analysis, as needed.
On days where only concentration analysis was required, the formulations were only sampled from the middle. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15% of theoretical concentration. For homogeneity, the criteria for acceptability were a relative standard deviation (RSD) of concentrations of ≤ 5% for each group.
Stability analysis: Stability analyses performed previously in conjunction with 1318012 demonstrated that the test substance is stable in the control substance when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability date are described in study 1318012.
Details on mating procedure:
Female rabbits were naturally bred at the Supplier by untreated breeder male rabbits of the same source and strain before shipment to the Testing Facility. The day mating occurred was designated as Gestation Day (DG) 0. The rabbits were shipped to the Testing Facility after mating, arriving on DG 1, 2, 3, 4 or 5.
Duration of treatment / exposure:
From DG 6 through DG 28
Female rabbits were administered the test substance or the control substance formulations by oral gavage (stomach tube). Doses were adjusted based on the most recently recorded body weight. The formulations were stirred continuously during dose administration.
Frequency of treatment:
once daily at approximately the same time each day.
Duration of test:
22 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control - Group 1
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
Group 4
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 5
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- The test system was selected because: 1) it is a standard species accepted for use in embryo-fetal development studies; 2) this species and strain has been demonstrated to be sensitive to developmental toxicants; and 3) historical data and experience exist at the Testing Facility.
- The number of rabbits chosen for this study was the smallest number considered necessary to provide the minimum number of pregnancies recommended by the applicable guidelines.
- Route rationale: The oral (gavage) route of exposure was selected because this is the intended route of human exposure.
- Dose selection rationale: Dose level selections were based on the results of the range-finding study. Mortality, adverse clinical signs, reduced body weight gain and food consumption were observed at 250 and 500 mg/kg/day. Abortions, reduced body weight gain and reduced food consumptions were also observed at 125 mg/kg/day.

Maternal examinations:
VIABILITY CHECKS (mortality):
- Time schedule: the rabbits were assessed for viability at least twice daily during the study

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The rabbits were observed for general appearance on the day of arrival at the Testing Facility and daily during the dose and post-dose period, including the day of scheduled euthanasia.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 1 to 2 hours after dose administration

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on DG 0 (provided by the Supplier), on the day of arrival at the Testing Facility, and daily during the dose and postdose period, including the day of scheduled euthanasia.

FOOD CONSUMPTION : Yes
- Food consumption values were recorded daily during acclimation (values not tabulated) and daily during the dose and post-dose periods including the day of euthanasia.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #: DG 29
- Method of euthanasia: A euthanasia solution (390 mg/mL) was used to euthanize rabbits (via intravenous injection)
Unscheduled Deaths: The rabbits that died or were euthanized before scheduled termination were examined for the cause of death or condition as soon as possible after the observation was made. The rabbits were examined for gross lesions and tissues identified were collected and retained for possible future evaluation. Pregnancy status and uterine contents were recorded, and the aborted fetuses, conceptuses in utero and/or delivered kits were examined to the extent possible, using the same methods described for term fetuses.
Scheduled euthanasia: To minimize bias, all Caesarean-sections, gross necropsies and fetal examinations were conducted without knowledge of dose group. On DG 29, surviving rabbits were euthanized, and examined for ovarian and uterine contents and gross lesion. Tissues identified were collected and retained for possible future evaluation.
- Necropsy: A gross necropsy of the thoracic, abdominal and pelvic viscera was performed for all rabbits. Images were generated for illustration of or consultation on gross observations. Generation of such images was documented. Images and associated documentation were retained and will be archived.
- Tissue collection and preservation: Representative samples of the organs examined (see below) were collected from all rabbits and preserved in 10% neutral buffered formalin, unless otherwise indicated. Unless specifically cited, all other tissues were discarded. Corresponding tissues were retained from one rabbit in the control group, in order to provide comparative tissues for any possible histopathological evaluations of gross lesions.
- Organs examined: cervix, esophagus, gravid uterus, gross lesions/ masses, heart, kidney, liver, lung, ovaries, spleen, stomach, trachea, and uterus.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes, the reproductive tract was dissected from the abdominal cavity
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: placentae (size, color, and shape), live and dead fetuses
A live fetus was defined as one that responds to stimuli. A dead fetus was defined as a term fetus that did not respond to stimuli and that was not marked autolyzed. Any dead fetuses demonstrating marked to extreme autolysis were considered to be late resorptions. A conceptus was defined as a late resorption if it was grossly evident that organogenesis had occurred; if that was not the case, the conceptus was defined as an early resorption.
Fetal examinations:
- Method of euthanasia: A euthanasia solution (390 mg/mL) was used to euthanize live fetuses (via intraperitoneal injection)
- External examinations: Yes, fetuses were examined for external abnormalities. Late resorptions and dead fetuses were examined for external abnormalities and sex to the extent possible. During the visceral examination, each fetus was internally sexed and examined for signs of altered reproductive development and/or cryptorchidism.
- Soft tissue examinations: Yes, fetuses in each litter were examined for visceral abnormalities by using a modification of the microdissection technique of Staples. A single cross-section was made between the parietal and frontal bones (i.e., coronal section), and the brain was examined in situ. After examination, each fetus was appropriately processed and the carcass was fixed in alcohol
- Skeletal examinations: Yes, each fetus was examined for skeletal abnormalities after staining with alizarin red S. Following examination, skeletal preparations were retained in glycerin with thymol added as a preservative.
- Head examinations: Yes
Statistics:
Any data collected during the pre-dose period were not tabulated or summarized. All statistical analyses were performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions were summarized as indicated below according to sex and occasion or by litter.
- Descriptive Statistical Analyses: Means, standard deviations, percentages, numbers, ratios and/or incidences were reported, as appropriate by dataset.
- Inferential Statistical Methods: All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted.
- Parametric/Non-parametric: Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
- Non-Parametric: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the pairwise comparisons were conducted using Dunn’s test.
- Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.
Indices:
Parental indices:
Pregnancy Rate = (Number of Animals Pregnant x100)/ Number of Animals Paired
Pre-Implantation Loss = (Number of Corpora Lutea - Number of Implants) x 100/ Number of Corpora Lutea
Post- Implantation Loss = (Number of Implants - Number of Live Fetuses) x 100/ Number of Implants
Sex ratio (%males) = Number Male Fetuses x 100 /Total Number of Fetuses
Litter % of Fetuses with abnormalities = Number of Fetuses in Litter with a given Finding x 100 / Number of Fetuses in Litter examined
Historical control data:
Available
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 100 mg/kg/day, there was an increase in the number of rabbits observed with fur loss and thin fur cover during the study period in comparison with the control group values. All additional clinical signs were considered unrelated to administration of the test substance because the observations noted were not dose dependent and/or the clinical signs were observed in one or two rabbits in the dose group. These clinical signs included ungroomed fur, staining of the fur, a scab on the skin, thin condition, sneezing, deformed nose and red liquid material and red aborted material present.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Three females at 100 mg/kg/day aborted and were euthanized during the study period. Due to the increased incidence in abortions being observed at 100 mg/kg/day, these abortions were considered to be test substance related.
One female at 50 mg/kg/day began delivering and was euthanized on the day of scheduled euthanasia. This was not considered to be test substance related because it was a single occurrence within this dose group.
One female at 75 mg/kg/day was found dead on Day 10 of Gestation (DG 10). Based on the necropsy findings, this death was attributed to an intubation error.
All additional female rabbits survived until scheduled euthanasia.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 100 mg/kg/day, there were statistically significant reductions (p ≤0.01) in body weight gain observed on DGs 24 to 29 and during the overall study period (DGs 6 to 29) in comparison with the control group values. Mean body weights on DG 29 were 99%, 98%, 99% and 95% of the control group value at 25, 50, 75 and 100 mg/kg/day, respectively. At 50 and 75 mg/kg/day, there were statistically significant reductions (p ≤0.01 or p ≤0.05) in body weight gain observed on DGs 24 to 29 in comparison with the control group value. These reductions were not considered to be test substance related because: 1) they were not dose dependent; and 2) they were single occurrences and did not impact the overall body weight gain for these dose groups during the overall study period. (see attachments for detailed data tables and historical control data)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 100 mg/kg/day, there were statistically significant reductions (p ≤0.01 or p ≤0.05) in food consumption observed on DGs 26 to 27, 27 to 28, 28 to 29 and 24 to 29 in comparison with the control group values. There were no additional test item-related changes in food consumption observed during this study. There were statistically significant reductions (p ≤0.01 or p ≤0.05) in food consumption observed on DGs 26 to 27 and 28 to 29 at 50 mg/kg/day in comparison with the control group values. These reductions were not considered to be test substance related because they were not dose dependent (see attachments for detailed data tables and historical control data).
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:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related maternal gross necropsy observations at doses up to and including 100 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
Three females at 100 mg/kg/day aborted and were euthanized during the study period. Due to the increased incidence in abortions being observed at 100 mg/kg/day, these abortions were considered to be test substance related. One female at 50 mg/kg/day began delivering and was euthanized on the day of scheduled euthanasia. This was not considered to be test substance related because it was a single occurrence within this dose group.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The litter averages for corpora lutea, implantations, percent pre-implantation loss, and percent post implantation loss were comparable among the five dose groups and did not significantly differ.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No dam that survived until scheduled euthanasia had a litter consisting of only resorbed conceptuses.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The litter averages for total number of resorptions, number of early or late resorptions, were comparable among the five dose groups and did not significantly differ.
Dead fetuses:
no effects observed
Description (incidence and severity):
The litter averages for number of live and dead fetuses were comparable among the five dose groups and did not significantly differ.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
Pregnancy occurred in 23 (95.8%), 22 (91.7%), 21 (87.5%), 23 (95.8%) and 24 (100%) rabbits at 0, 25, 50, 75 and 100 mg/kg/day, respectively. Reflecting the rabbits at 50, 75 and 100 mg/kg/day that delivered, aborted or were found dead, as described previously, Caesarean-sectioning observations were based on 23, 22, 20, 22 and 21 pregnant rabbits in Groups 1 through 5, respectively.
Other effects:
not specified
Details on maternal toxic effects:
Three females at 100 mg/kg/day aborted and were euthanized during the study period. Due to the increased incidence in abortions being observed at 100 mg/kg/day, these abortions were considered to be test substance related. One female at 50 mg/kg/day began delivering and was euthanized on the day of scheduled euthanasia. This was not considered to be test substance related because it was a single occurence within this dose group. One female at 75 mg/kg/day was found dead on Gestation Day (DG) 10. This female had dark red fluid in the thoracic cavity and the cranial and caudal lobes of the lungs appeared dark red. Based on the necropsy findings, this death was attributed to an intubation error.
At 100 mg/kg/day, there was an increase in the number of rabbits observed with fur loss and thin fur cover during the study period in comparison with the control group values.
At 100 mg/kg/day, there were statistically significant reductions in body weight gain observed on DGs 24 to 29 and during the overall study period (DGs 6 to 29) in comparison with the control group values.
At 100 mg/kg/day, there were statistically significant reductions in food consumption observed on DGs 26 to 27, 27 to 28, 28 to 29 and 24 to 29.
There were no test substance-related maternal gross necropsy observations at doses up to and including 100 mg/kg/day.
Pregnancy occurred in 23 (95.8%), 22 (91.7%), 21 (87.5%), 23 (95.8%) and 24 (100%) rabbits at 0, 25, 50, 75 and 100 mg/kg/day, respectively. Reflecting the rabbits at 50, 75 and 100 mg/kg/day that delivered, aborted or were found dead, as described previously, Caesarian-sectioning observations were based on 23, 22, 20, 22 and 21 pregnant rabbits in groups 1 through 5, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
mortality
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 100 mg/kg/day, there was a 7% reduction in fetal body weights (both) in comparison with the control group value. There was a 8% reduction in gravid uterus weight in the females at 100 mg/kg/day in comparison with the control group values.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The litter averages for percent live male and female fetuses, number of live male and female fetuses were comparable among the five dose groups and did not significantly differ.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No test substance-related fetal external alterations were observed on study. Single fetuses at 50 and 100 mg/kg/day had hyperflexion in the right forepaw. One fetus at 75 mg/kg/day had hyperextension in the left forepaw. One fetus at 100 mg/kg/day had adactyly on both forepaws. These malformations were not considered to be test substance related because they were single occurrences within each of the dose groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 mg/kg/day, there was an increased incidence of unossified pubes in the pelvic girdle as compared to the control group value. The increases were not considered toxicologically important because the frequency was within the spontaneous alteration reference data range for the New Zealand White rabbit (Stadler et al., 1983).
There were no additional skeletal alterations that were considered test substance related because the findings were not dose-dependent, the number of affected litters/fetuses were similar to controls, and/or number of affected litters/fetuses were within the expected historical incidence (see Historical Control Data table below). Skeletal alterations consisted of common skeletal variations including abnormalities of the forelimb (absent metacarpal and phalanges); abnormalities of the ribs (branched, interrupted, nodulated and shortened); abnormalities of the skull (incomplete ossification of the frontals, a bent right hyoid ala, incomplete ossification of the interparietal, a hole in the parietal(s) and supernumerary suture bone) abnormalities of the sternebrae (fused, misshapen, unossified, incompletely ossified and isolated ossification site); supernumerary ribs (short); and abnormalities of the vertebrae (misaligned, unossified or incomplete ossification of the caudal vertebra; absent or misshapened cervical arch; unossified or incomplete ossification of the cervical centrum; incomplete ossification of the sacral centrum; and misaligned, unossified or incomplete ossification of the thoracic centrum; and a hemivertebra in the thoracic vertebra). See attachments for detailed data tables and historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
All visceral abnormalities (malformations and variations) that were observed were considered unrelated to the test substance because the findings were not dose-dependent, the number of affected litters/fetuses were similar to controls, and/or number of affected litters/fetuses were within the expected historical incidence. These abnormalities included an interrupted aortic arch, circumcorneal hemorrhage in the right or left eye, persistent truncus arteriosis, muscular ventricular septal defect, membranous ventricular septal defect, thin ventricular wall on the right side of the heart, absent innominate artery, malpositioned right kidney, discolored spleen, small spleen, retroesophageal right subclavian artery and malpositioned right subclavian artery.
Other effects:
no effects observed
Description (incidence and severity):
Fetal Ossification Site Averages: There were no differences among the control and the dose groups administered the test substance in the average numbers of ossification sites per fetus for the hyoid, vertebrae (cervical, thoracic, lumbar, sacral and caudal), ribs, sternum (manubrium, sternal centers and xiphoid), forelimbs (carpals, metacarpals and phalanges) or hind limbs (tarsals, metatarsals and phalanges).
Details on embryotoxic / teratogenic effects:
At 100 mg/kg/day, there was a 7% reduction in fetal body weights (both) in comparison with the control group value. There was also a 5% reduction in terminal body weight and an 8% reduction in gravid uterus weight in the females at 100 mg/kg/day in comparison with the control group values. There was a low incidence of fetal skeletal abnormalities noted in the 100 mg/kg/day dose group. The only fetal skeletal malformation that was attributed to the test substance was an increased incidence of unossified pubes in the pelvic girdle which were noted in 6 fetuses from 3 different litters within the 100 mg/kg/day dose group. This is considered as within the spontaneous malformation reference data range (Stadler et al, 1983). Moreover, the only skeletal finding is isolated and not associated with any other treatment-related skeletal abnormalities or external/visceral abnormalities. There were no test substance-related differences noted in the gross external abnormalities, soft tissue abnormalities and fetal ossification site.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Dose formulation analyses:

Dose formulations prepared in RO deionized water were analyzed following the first and last preparations. The dose formulations were measured, and the mean concentrations were found to be within protocol specification of +/-10% (individual values within +/-15%) of their theoretical concentrations of 5, 10, 15 and 20 mg/mL. The first preparation of the dosing suspensions of 5, 10, 15 and 20 mg/mL were analyzed and found to be 105%, 108%, 107% and 109% of the target concentrations, respectively. The back-up samples for the 20 mg/mL samples were analyzed and the results were within the acceptance criteria. The homogeneity values obtained for the first preparation was 3.6%, 0.64%, 0.95% and 3.5% RSD for the 5, 10, 15 and 20 mg/mL concentrations, respectively. The last preparation of the dosing suspensions of 5, 10, 15 and 20 mg/mL were analyzed and found to be 109%, 95%, 103% and 104% of the target concentrations, respectively.

Data tables:

Conclusions:
The test substance [0 (vehicle control), 25, 50, 75 or 100 mg/kg/day] was administered by oral gavage to pregnant rabbits from DGs 6 to 28. At 100 mg/kg/day, an increased number of abortions, an increased incidence in fur loss and thin fur cover, and a decrease in body weight gain and food consumption during the study period. There was a reduction in gravid uterine weights and fetal body weights at the time of Caesarean-sectioning. Fetal skeletal alterations occurred at increased incidences in the 100 mg/kg/day dose group. These alterations were isolated and within the spontaneous malformation reference data range for the New Zealand White rabbit (Stadler et al., 1983). The maternal no observed adverse effect level (NOAEL) for the test substance is 75 mg/kg/day and the developmental NOAEL is 100 mg/kg/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Prenatal development toxicity study in rabbit (OECD guideline 414)

Female rabbits were administered the test substance (25, 50, 75 and 100 mg/kg/day) or the control substance formulations by oral gavage (stomach tube) once daily on day 6 through 28 of preumed gestation (DGs 6 through 28). Doses were adjusted based on the most recently recorded body weight and administered at approximately the same time each day. The formulations were stirred continuously during dose administration. The following parameters and endpoints were evaluated in this study: maternal viability, maternal clinical signs, maternal body weights, maternal body weight changes, and maternal food consumption, gross necropsy findings, ovarian and uterine contents, and fetal examinations. Three females at 100 mg/kg/day aborted and were euthanized during the study period. Due to the increased incidence in abortions being observed at 100 mg/kg/day, these abortions were considered to be test substance related. One female at 50 mg/kg/day began delivering and was euthanized on the day of scheduled euthanasia. This was not considered to be test substance related because it was a single occurrence within this dose group. One female at 75 mg/kg/day was found dead on gestation day (DG) 10. This female had dark red fluid in the thoracic cavity and the cranial and caudal lobes of the lungs appeared dark red. Based on the necropsy findings, this death was attributed to an intubation error.

At 100 mg/kg/day, there was an increase in the number of rabbits observed with fur loss and thin fur cover during the study period in comparison with the control group values.

At 100 mg/kg/day, there were statistically significant reductions in body weight gain observed on DGs 24 to 29 and during the overall study period (DGs 6 to 29) in comparison with the control group values.

At 100 mg/kg/day, there were statistically significant reductions in food consumption observed on DGs 26 to 27, 27 to 28, 28 to 29 and 24 to 29.

There were no test substance-related maternal gross necropsy observations at doses up to and including 100 mg/kg/day.

Pregnancy occurred in 23 (95.8%), 22 (91.7%), 21 (87.5%), 23 (95.8%) and 24 (100%) rabbits 0, 25, 50, 75 and 100 mg/kg/day, respectively. Reflecting the rabbits at 50, 75 and 100 mg/kg/day that delivered, aborted or were found dead, Caesarian-sectioning observations were based on 23, 22, 20, 22 and 21 pregnant rabbits in groups 1 through 5, respectively. At 100 mg/kg/day, there was a 7% reduction in fetal body weights (both) in comparison with the control group value. There was also a 5% reduction in terminal body weight and an 8% reduction in gravid uterus weight in the females at 100 mg/kg/day in comparison with the control group values.

There was a low incidence of fetal skeletal abnormalities noted in the 100 mg/kg/day dose group. The only fetal skeleral malformation that was attributed to the test substance was an inreased incidence of unossified pubes in the pelvid girdle which were noted in 6 fetuses from 3 different litters within the 100 mg/kg/day dose group. It is considered as within the spontaneous malformation reference data range (Stadler et al., 1983). Moreover, the only skeletal finding is isolated and not associated with any other treatment-related skeletal abnormalities or external/visceral abnormalities. There were no test substance-related differences noted in the gross external abnormalities, soft tissue abnormalities and fetal ossification site.

In conclusion, the test substance was administered by oral gavage to pregnant rabbits from DGs 6 to 28. At 100 mg/kg/day, an increased number of abortions, an increased incidence in fur loss and thin fur cover, and a decrease in body weight gain and food consumtion during the study period. There was a reduction in gravid uterine weights and fetal body weights at the time of caesarian-sectioning. Fetal skeletal abnormalities occurred at increased incidences in the 100 mg/kg/day dose group is isolated and considered as within the spontaneous malformation reference data range (Stadler et al., 1983). The maternal NOAEL is considered to be 75 mg/kg/day while the developmental NOAEL is considered to be 100 mg/kg/day.

Prenatal development toxicity study in rat (OECD guideline 414)

Based on the results in the prenatal developmental toxicity study, the maternal No Observed Adverse Effect Level (NOAEL) for the substance was established as being 450 mg/kg/day, based on reduced body weights and body weight gain of the high dose animals at 1000 mg/kg/day. In the absence of treatment-related effects on reproductive parameters, fetal body weight and fetal morphology, a dosage level of 1000 mg/kg/day was considered to be the NOAEL for developmental toxicity.

However, in the RAC Opinion proposing harmonised classification and labelling at EU level of the test substance, adopted 9 December 2016 (CLH-O-0000001412-86-132/F), is was considered that in this stuy,  delayed postnatal development was observed in the 2-generation study, expressed as a statistically significant delay in balanopreputial separation and vaginal opening in the F1 generation. The anogenital distance was significantly lower in males at 150 and 450 mg/kg bw/d and in females at 150 mg/kg bw/d in the F2 generation. A classification for developmental toxicity in category 2 was therefore proposed (Repr. 2, H361d).

Toxicity to reproduction: other studies

Description of key information

No other studies available

Justification for classification or non-classification

No developmental toxicity occurred in the studies performed up to and including the highest test concentration. Furthermore, the effects seen in the two generation reproduction study at dose level of 1000 mg/kg bw/day do not provide sufficient evidence for classificiation under CLP. However, based on the RAC opinion and inclusion in Annex VI of the CLP regulation (13th ATP), the substnace is classified as Repr. 2, H361fd.

Additional information