Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 July 2016 - 12 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
A total of 90 Hsd Sprague Dawley SD rats (45 males and 45 virgin females), 6 to 7 weeks old and weighing 175 to 200 g for males and 151 to 175 g for females, were ordered from and supplied by Envigo RMS srl, San Pietro al Natisone (UD), Italy.
After arrival, on 28 July 2016, the weight range for each sex (192-202 g for males, 164-173 g for females, slightly outside the range at order for the males) 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 20 days 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. Animal room controls were set to maintain temperature and relative humidity at 22 °C±2 °C and 40-70% respectively; actual conditions were monitored, recorded and the records retained. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
The required amount of the test substance was dissolved in the vehicle. The formulations were prepared weekly during the first 5 weeks of the study and daily thereafter up to the end of the study (concentrations of 6.25, 25 and 100 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 the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
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 of copulation (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:
The analytical method was validated in RTC Study No. A2213 in the range from 5 to 125 mg/mL. Linearity, accuracy and precision were within the limits stated in RTC SOPs for solutions (r > 0.98; accuracy 95-105 %; precision CV < 5 %).
Stability was verified in the range from 5 to 125 mg/mL and was found to be at least 24 hours at room temperature and 8 days at 5±3°C.
The proposed formulation procedure for the test substance was checked in the range from 5 to 125 mg/mL by chemical analysis (concentration) in RTC Study no. A2213 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (90-110 %).
Samples of the formulations prepared on Day 1 and Week 6 were analysed to check the concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for concentration of solutions (90-110 %).
Duration of treatment / exposure:
- Males: Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and up to the day before necropsy.
- Females: Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods up to Day 3 post partum.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
Each group comprised 10 male and 10 female rats (Group 1 to 4).

Examinations

Parental animals: Observations and examinations:
1) Clinical signs: 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.

2) Observations of the cage tray: observations of the cage tray were performed during mating period for all main groups and were recorded daily.

3) Body weight: males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to 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.
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 cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Sperm parameters (parental animals):
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.

Litter observations:
A parturition check was performed from Day 20 to Day 25 post coitum. Female nos. 3 and 9 (Group 1), nos. 21 and 37 (Group 2) and no. 65 (Group 4), which did not give birth after 25 days of post coitum period, were sacrificed shortly after (Day 26 post coitum). These animals were found not pregnant at necropsy. Gestation periods were taken as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.
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. Observations were performed once daily for all litters.
Postmortem examinations (parental animals):
Parental animals were killed by exsanguination under isofluorane anaesthesia.

1) Parental males: the males were sacrificed after completion of the mating period after 36 days of treatment.

2) Parenteral females: the females with live pups were sacrificed on Day 4 post partum. The females which did not give birth 25 days after positive identification of mating were killed shortly after.

3) Necropsy: The clinical history of the animals 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
- Females: 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 10-20% solution of ammonium sulphide to reveal evidence of implantation.

4) Organ weights: from all animals completing the scheduled test period, the following organs were dissected free of fat and weighed: adrenal glands, brain (cerebrum, cerebellum, medulla/pons), epididymides, kidneys, liver, ovaries, prostate glands, seminal vescicles with coagulating glands, testes, thyroid, uterus including cervix. The ratios of organ weight to body weight were calculated for each animal.

5) Tissues fixed and preserved: Samples of all the following tissues 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): abnormalities, adrenal glands, brain (cerebrum, cerebellum, medulla/pons), clitoral gland, epididymides, kidneys, liver, ovaries, penis with preputial gland, prostate gland, seminal vesicles with coagulating glands, spleen, testes, thymus, thyroid, uterus – cervix, vagina.

After dehydration and embedding in paraffin wax, sections of the 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.
The examination was restricted as follows:
- tissues specified above from from all animals in the control and high dose group killed at term
- all abnormalities in all groups.
Postmortem examinations (offspring):
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 confirmation 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. Inter group differences between the control and treated groups were assessed by the non-parametric version of theWilliams 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 if ‘n’ was more than 5.
Reproductive indices:
Group mean values were calculated for all parameters. Data for not pregnant females were excluded from means. The following reproductive indices were calculated:

1) Males:
- Copulatory Index (%) = no. of animals mated x 100/ no. of animals paired
- Fertility Index (%) = no. of males which induced pregnancy x 100/ no. of males paired

2) Females:
- Copulatory Index (%) = no. of animals mated x 100/ no. of animals paired
- Fertility Index (%) = no. of pregnant females x 100/ no. of females paired
Offspring viability indices:
1) Males and females:
Pre coital Interval = Mean number of days between pairing and mating

2) Females:
Pre-birth loss was calculated as a percentage from the formula:
no. of visible implantations − total litter size at birth / no.of visible implantations ×100

Pre-implantation loss was calculated as a percentage from the formula:
no. of corpora lutea − no. of visible implantations / no. of corpora lutea ×100

Pup loss at birth was calculated as a percentage from the formula:
Total litter size at birth − live litter size at birth / Total litter size at birth ×100

Cumulative 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 ×100

Sex 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:
no effects observed
Description (incidence and severity):
Slight to marked red staining in the cage tray and slight to moderate red faeces were observed during the pre-mating, post mating / gestation and post partum phases in cages of mid - and high dose animals.
Slight to marked red staining in the cage tray and moderate red faeces were observed during the mating phase in some cages of the low dose group animals and in all cages of mid - and high dose animals.
The above observations were considered due to excretion of metabolised test item (data not tabulated).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment - related changes were noted. The sporadic lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.
Spermatogenic cycle: 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.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No treatment - related anomalies were noted in the oestrous cycle of the treated females, when compared to controls. The inter - group differences observed in the mean pre - coital interval were not dose - related and, therefore, they were considered to be incidental. All females mated.
Male no. 44 (Group 3) did not mate with female no. 43, which subsequently mated with male no. 42 of the same group.
Males: the copulatory index was 100% for Groups 1, 2 and 4 and 90% for Group 3. The fertility index was 80% for Groups 1 and 2, 100% for Group 3 and 90% for Group 4.
Females: the copulatory index was 100% for all groups. The fertility index was 80% for Groups 1 and 2, 100% for Group 3 and 90% for Group 4.

Details on results (P0)

No effects on sexual function and fertility or in developmental parameters and lactation were observed at any of the dose levels investigated.
No treatment - related anomalies were noted in the oestrous cycle of the treated females, when compared to controls. Copulatory and fertility indices did not show any treatment - related differences among treated and control groups.

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 and a regular layering in the germinal epithelium was described.

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related systemic toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No significant differences in total litter size, live litter size, mean pup loss, sex ratio and mean pup weights were observed among the treated dams and the controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Autolysed abdominal and / or thoracic organs were generally observed in pups found dead at birth and in pups which died during the lactation period.
Histopathological findings:
no effects observed
Other effects:
not examined
Description (incidence and severity):
Parturition, lactation, implantation, litter data and sex ratio did not show any changes of toxicological relevance.

Necropsy findings in pups did not reveal any treatment - related effect. Furthermore, no relevant changes were detected at post mortem examination in treated parental animals, when compared with controls.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect on development

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the NOEL (No Observed Effect Level) of the substance for systemic toxicity and for fertility and reproduction parameters was considered to be 1000 mg/kg bw/d in rats.
Executive summary:

A study was conducted to determine the reproductive and developmental toxicity of the substance according to OECD Guideline 421, in compliance with GLP. The test substance was administered daily by oral gavage to male (36 d) and female (from 41 to 59 d) Sprague Dawley SD rats at dose levels of 0, 62.5, 250 and 1000 mg/kg bw/d. The following investigations were performed on parental animals of all groups: mortality check, clinical signs, body weight, food consumption, oestrous cycle, mating performance, litter data, macroscopic observations and organ weights. Measurement of body weight, clinical signs and macroscopic observations of pups were also performed. Histopathological evaluation of reproductive organs/tissues was performed on all control and high dose males and females, as well as on all abnormalities detected during post mortem observation. The identification of the stages of the spermatogenic cycle was performed in all males of the control and high dose groups. As a result of the treatment no systemic and reproductive/developmental toxicity was observed in the parental animals as well as no signs of developmental toxicity in the pups. Under the study conditions, the NOEL of the substance for systemic toxicity and for fertility and reproduction parameters was considered to be 1000 mg/kg bw/d (Longobardi, 2017).