Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
other: read across from similar substance
Adequacy of study:
key study
Study period:
2016-06-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
BY29 - similar substance 01
IUPAC Name:
BY29 - similar substance 01

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
A total of 90 Hsd: Sprague Dawley SD rats (45 males and 45 virgin females), 8 to 9 weeks old and weighing 225.6-253.2 g for males and 200-215.7 g for females, were received from Harlan Italy S.r.l., San Pietro al Natisone (UD), Italy. After arrival the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian. An acclimatisation period of approximately 2 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations. The animals were housed in a limited access rodent facility. From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5 x 38 x 20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polycarbonate cages measuring 42.5 x 26.6 x 18 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages measuring 42.5 x 26.6 x 18.5 cm for the gestation period, birth and lactation. Suitable nesting material was provided and changed as necessary. Nesting material was changed at least 2 times a week.Animal room controls were set to maintain temperature and relative humidity at 22 °C +/-2 °C and 55% +/- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day. Drinking water was supplied ad libitum to each cage via water bottles. A commercially available laboratory rodent diet was offered ad libitum throughout the study.In-life phase: 03 June-08 August 2015 (last day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% aqueous solution
Details on exposure:
The required amount of the test item was suspended in the vehicle (0.5% aqueous solution of carboxymethylcellulose). The formulations were prepared daily (concentrations of 3, 5, 9.5 and 20 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied. The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at thesame dose volume.
Details on mating procedure:
Matings were monogamous (one male to one female). Vaginal smears were taken from the day after the start of pairing until positive identification ofcopulation (sperm identification, vaginal plug in situ or copulation plug found on the cage tray). Each cage tray was checked each morning for the presence of copulation plugs. The female was paired with the same male until positive identification of copulation occurred or 14 days had elapsed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the formulations, prepared on Week 1 and last week of treatment, were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for suspensions (80-120% for concentration and CV < 10% for homogeneity).Chemical analysis was carried out by the Analytical Chemistry Department at RTC using a validated method (RTC Study no. A0145).The software used for this activity was Empower® Probuild No. 2154.
Duration of treatment / exposure:
Males:Animals were dosed for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy, for at least 28 consecutive days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.Females:Animals were dosed for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for each animal according to the last recorded body weight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Frequency of treatment:
once daily, 7 days a week
Details on study schedule:
in concordance with OECD 421
Doses / concentrations
Remarks:
Doses / Concentrations:0, 30, 50, 95, 200Basis:nominal conc.
No. of animals per sex per dose:
Each group comprised 10 male and 10 female rats. The 50 mg/kg bw/day dose group comprised 5 male and 5 female rats.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:Dose levels were selected in consultation with the Sponsor based on a 28-day oral repeat-dose study (RTC Study no. A0156) conducted with dose levels of 300/200, 95, and 30 mg/kg body weight/day. Based on this study dose levels of 200 (dose group 4), 95 (dose group 3), and 30 (dose group 2) mg/kg body weight/day were selected for the developmental screening study. Mortality was noted in high dose group (dose group 4) during the first 2 days of dosing. The selected dosage of 200 mg/kg body weight/day was considered not suitable for the study and for this reason animals were sacrificed after 2 days of treatment. The former medium dose group of 95 mg/kg body weight/day was defined as high-dose group and a new medium-dose group of 50 mg/kg body weight/day (dose group 5) was added to the study. Control animals are belonging to dose group 1.
Positive control:
N.A.

Examinations

Parental animals: Observations and examinations:
MortalityThroughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.Clinical signsAll clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions (1-1.5 hour after treatment).Body weightMales were weighed on the day of allocation to treatment groups, on the day of treatment commence, weekly thereafter and at termination.Females were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter until positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.Food consumptionThe weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period following allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum, starting from Day 1 post partum. The measurement of food intake for Group 5 animals started on Day 1 of treatment.
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting from two weeks before pairing until a positive identification of copulation was made.The vaginal smear data were examined to determine the following:1. anomalies of the oestrous cycle2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Sperm parameters (parental animals):
Parameters examined in male parental generations:testis weight, epididymis weight. The testis and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS).
Litter observations:
A parturition check was performed from Day 20 to Day 25 post coitum.As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and only live pups were identified. Live pups were individually weighed on Days 1 and 4 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy. These observations were performed once daily for all litters.
Postmortem examinations (parental animals):
EuthanasiaParental animals were killed by exsanguination under isofluorane anaesthesia. All animals were subjected to necropsy, supervised by a pathologist. The surviving males and females of Group 4 were sacrificed after the interruption of the treatment on Day 3 of the study.Groups 1, 2 and 3 males were killed after 34 days of treatment. Males of Group 5 were killed after 29 days treatment.Parenteral females with live pups were sacrificed on Day 4 post partum. The females showing no evidence of copulation were killed after 25 days of the last day of the mating session. The females which did not give birth 25 days after positive identification of mating were killed shortly after.NecropsyNo necropsy was performed, no terminal body weight was collected and no organs were weighed for Group 4 animals sacrificed on Day 3 of the study.For all other animals, the clinical history was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.All females were examined also for the following:– external and internal abnormalities;– number of visible implantation sites (pregnant animals);– number of corpora lutea (pregnant animals).Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.PupsAll pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination (Day 4 post partum) were killed and examined for external abnormalities and sex confirmed by gonadal inspection.Organ weightsFrom all animals completing the scheduled test period, the organs required were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.Tissues fixed and preservedSamples of all the tissues (all parental animals)required were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol).Histopathological examinationAfter dehydration and embedding in paraffin wax, sections of the required tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed for Groups 1 and 3.The examination was restricted as detailed below:i) Required tissues from all animals of Groups 1 and 3ii) All abnormalities in all groups
Postmortem examinations (offspring):
Pups were sacrificed by intraperitoneal injection of Sodium Thiopental. All pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination (Day 4 post partum) were killed and examined for external abnormalities and sex confirmed by gonadal inspection.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test.
Reproductive indices:
MalesCopulation Index (%) =no. of males mated x 100/no. of males pairedFertility Index (%) = no. of males which induced pregnancy x 100/no. of animals pairedFemalesCopulatory Index (%) = no. of females mated x 100/no. of females pairedFertility Index (%) = no. of pregnant females x 100/no. of females paired
Offspring viability indices:
Males and femalesPre coital Interval = Mean number of days between pairing and matingFemalesPre-birth loss was calculated as a percentage from the formula:no: of visible implantations - total litter size at birth/no: of visible implantations x 100Pre-implantation loss was calculated as a percentage from the formula:no: of corpora lutea - no: of visible implantations/no: of corpora lutea x 100Pup loss at birth was calculated as a percentage from the formula:Total litter size - live litter size/Total litter size x 100Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:Total litter size at birth - live litter size at Day 4/Total litter size at birth x 100Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No signs after reduction of the high dose level
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Mortality and fate of females:Two females of Group 4, receiving 200 mg/kg body weight, died on Day 1 of the study after a single administration. On Day 2 of dosing, 1 male and 1 female were found dead and two females were sacrificed for humane reasons. The selected dose of 200 mg/kg body weight/day was therefore considered not suitable for the study and treatment was stopped. All surviving animals in the group were sacrificed on Day 3 of the study. The study was conducted using 2 treatment groups and 1 control group each of 10 males and 10 females (Groups 1, 2 and 3) and 1 additional treated group comprising 5 males and 5 females (Group 5). One control female and two females of Group 3 did not mate. A total of 3 females in the control group and 4 females in Group 3 were found not pregnant at necropsy. The number of females with live pups at Day 4 post partum were 7 in the control group, 10 in the low dose group, 5 in the mid-dose group and 6 in the high dose group.Clinical signs:At daily clinical observation, no signs were noted in males during the entire treatment period.Single occasions of rales and staining of the fur in different females of Group 3 were described and considered incidental. No other signs were observed.Body weight and body weight gain: Body weight was unaffected by treatment in both sexes. A slight but significant reduction in body weight gain was noted in treated males receiving 50 (Group 5) and 95 (Group 3) mg/kg body weight on Day 8 of treatment, but the animals recovered thereafter. No toxicological relevance was attributed to the slight increase in body weight gain noted in pregnant females receiving 30 mg/kg body weight (Group 2) on Day 14 post coitum, since it was considered due to the good pregnancy status noted in this group (all females were pregnant).Food consumption:No differences in food consumption were observed in treated animals when compared to controls.Oestrous cycle, reproductive parameters, pairing combination and mating performance:A slight reduction in number of days with presence of oestrous cycle was noted in Groups 3 and 5 females. This change was not clearly related to treatment. Reproductive parameters and mating performance were comparable between groups.Implantation, pre-birth loss data and gestation length of females:Implantation, pre-birth loss data and gestation length were similar between groups.Terminal body weight and organ weights:Terminal body weight was slightly reduced in treated animals when compared to controls.Absolute organ weights were unaffected by treatment for both sexes. The slight increase in relative weight of adrenals, epididymides and seminalvesicles in Group 3 or 5 males is considered due to the lower terminal body weight noted in these animals, when compared to controls.Macroscopic observations:No relevant changes were noted in treated animals. The sporadic changes observed in few treated animals could be considered incidental.Microscopic observations:No treatment-related changes were noted. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
95 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

Litter data, implantation, pre- and post-implantation losses and sex ratios did not differ between groups. Necropsy findings in pups did not show anyfindings related to treatment.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
95 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of the present study, the NOAEL was considered to be 95 mg/kg body weight/day for males, for females and offspring.
Executive summary:

In a reproduction/developmental toxicity screening test (OECD 421) the test item (94.4% purity) was adminstered orally to 10 male and female Sprague Dawley rats in 0.5% carboxy methylcellulose by gavage at dose levels of 0, 30, 95 and 200 mg/kg bw/day. Mortality was noted in 200 mg/kg bw/day dose group during the first 2 days of dosing. The selected dosage of 200 mg/kg body weight/day was considered not suitable for the study and for this reason animals were sacrificed after 2 days of treatment. The former medium dose group of 95 mg/kg body weight/day was defined as high-dose group and a new medium-dose group of 50 mg/kg body weight/day (dose group 5, five animals/sex) was added to the study. Male and female animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing. Thereafter, male animals were dosed through the day before necropsy, for at least 28 consecutive days. Female animals were dosed thereafter during pairing, post coitum and post partum periods until day 3 post partum or the day before sacrifice.

No relevant differences were observed in clinical signs, body weight, body weight gain and food consumption in treated animals compared to the controls. The reproductive parameters, except for a reduction in number of days with presence of oestrous cycle observed in females treated at 50 and 95mg/kg body weight/day with unclear relation to treatment, in terms of mating performance,

pre-coital interval and copulatory evidence, were similar between groups. Litter data, implantation, pre- and post-implantation losses and sex ratios did not differ between groups. Necropsy findings in pups did not show any findings related to treatment. Histopathological examination did not reveal any treatment-related effect. Based on the results of the present study, the NOAEL was considered to be 95 mg/kg body weight/day for males, for females and offspring.

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