Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
04 June 2012 - 27 August 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550 Reproduction/Developmental Toxicity Screening Test, July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Physical state: White powder
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI(Han)
- Age at study initiation: (P) approximately 11 weeks.
- Housing:
> Pre-mating: animals were housed in groups of 5 animals/sex/cage in plastic cages.
> Mating: females were caged together with males on a one-to-one-basis in plastic cages.
> Post-mating: males were housed in their home cages with a maximum of 5 animals/cage. Females were individually housed in plastic cages.
> Lactation: pups were kept with the dam until termination in plastic cages.
- Diet: Pelleted rodent diet, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24 °C
- Humidity (%): 40 – 70 %
- Air changes (per hr): 15 room air changes/hour.
- Photoperiod (hrs dark / hrs light): A 12 hour light/12 hours dark cycle was maintained.

IN-LIFE DATES: From: 04 June To: 02 August (males) 27 August (females and pups).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity/density of the vehicle. No correction was made for the purity/composition of the test material. Test solutions were stored at ambient temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the test facility.
- Specific gravity: 1.036
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. Detection of mating was not confirmed for animal no. 61 which did deliver live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on two occasions during the treatment phase Day 6 (09 July 2012) and Day 10 (13 July 2012) according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

- Analytical conditions:
Instrument: Alliance Separation Module 2695 (Waters, Milford, MA, USA)
Detector: Dual λ Absorbance Detector 2487 (Waters)
Column: Symmetry C-18, 150 mm × 3 mm i.d., dp = 5 μm (Waters)
Column temperature: 40°C ± 1°C
Injection volume: 100 μL
Mobile phase: 80/20 (v/v) acetonitrile/water
Flow: 1.0 mL/min
UV detection: 235 nm

- Sample Preparation
Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 25 mL (Day 6) or 50 mL (Day 10).
For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50% height and stored at room temperature under normal laboratory light conditions for 6 hours. The volumetric flasks were filled up to the mark and further diluted with acetonitrile. The last step was a dilution in a 1:1 (v/v) ratio with water to obtain a matrix of50/50 (v/v) acetonitrile/water and concentrations within the calibration range.

- Calibration Solutions
> Stock and spiking solutions: prepared in acetonitrile at concentrations of 935 and 1145 mg/L.
> Calibration solutions: prepared in the concentration range of 6.5 - 200 μg/L were prepared from two stock solutions. The end solution of the calibration solutions was 50/50 (v/v) acetonitrile/water.
> Procedural recovery samples: Approximately 500 mg blank vehicle was spiked with the test mateiral at a target concentration of 20 or 200 mg/L. The accuracy samples were treated similarly as the test samples.

- Sample injections: Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

- Specification: The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 days (2 weeks prior to mating, during mating, and up to termination). Females were exposed for 39-54 days (during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation).
Frequency of treatment:
Once daily, seven days per week, up to the day before shceduled necropsy.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Ten per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosing range for the definitive study was based on the results of a 10-day dose range finding study. Three female rats were dosed at 500 and 1000 mg/kg bw/day for 10 consecutive days.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.
- Cage side observations checked: Mortality/viability and clinical signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily within 30 minutes after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Calculated weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: No quantitative investigation was performed; however subjective appraisal was maintained during the study.

OTHER: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect
signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any
deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Sperm parameters (parental animals):
From the males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.
Litter observations:
MORTALITY/VIABILITY
The number of live and dead pups was recorded on Day 1 of lactation and daily thereafter.

CLINICAL SIGNS
Detailed clinical observations were conducted at least once daily for all animals.

BODY WEIGHTS
Live pups were weighed on Days 1 and 4 of lactation.

SEX
Sex was determined for all pups on Days 1 and 4 of lactation.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: All surviving animals which delivered a litter were scarified on lactation Day 5-7. All surviving animals which failed to deliver a litter were sacrificed approximately 21 days after the last day of the mating period (i.e. females without evidence of mating).
All spontaneous deaths were submitted for necropsy as soon as possible after death and always within 24 hours.

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination: Cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, vagina, and all gross lesions.

The following male organs were weighed: Epididymides and testes.
Postmortem examinations (offspring):
SACRIFICE
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.

GROSS EXAMINATION OF DEAD PUPS:
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (manyto-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
- Mating index (%):
(Number of females mated/Number of females paired) x 100

- Fertility index (%):
(Number of pregnant females/Number of females paired) x 100

- Conception index (%):
(Number of pregnant females/Number of females mated) x 100

- Gestation index (%):
(Number of females bearing live pups/Number of pregnant females) x 100

- 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 of lactation:
(Number of dead pups on Day 4 of lactation/ Number of live pups at First Litter Check) x 100

- Viability index:
(Number of live pups on Day 4 post partum/Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related effects.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment related effects.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment related effects.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment related effects.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related effects.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related effects.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No treatment related effects.
Reproductive performance:
no effects observed
Description (incidence and severity):
No treatment related effects.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred during the study period that was considered to be related to treatment with the test material.
One female (no. 67) at 300 mg/kg died during delivery. Subsequent examination could not determine a cause of death that was related to treatment. This was considered to be incidental in nature and not related to treatment.
No clinical signs of toxicity were noted during the observation period.
Alopecia was noted for a single female (no. 52) at 100 mg/kg. This was incidental in nature.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals up to 1000 mg/kg.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The spermatogenic staging profiles were normal for all males evaluated.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically relevant effects on reproductive parameters were noted. There were 10, 9, 9 and 10 pregnant females in the control, 100, 300 and 1000 mg/kg groups, respectively.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no toxicologically relevant effects on testes and epididymides weights and terminal body weights up to 1000 mg/kg.
The testes to body weight ratio was significantly lower for males at 300 mg/kg. This was mainly attributable to the low ratio for one animal (no. 28) and was not considered to be related to treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Necropsy did not reveal any alterations that were attributable to treatment up to 1000 mg/kg.
The female (no. 67) who died during delivery was noted with dilation and a prolapsed vagina, a greenish, soft nodule on the right clitoral gland and 2 fetuses were found in the right uterine horn.
Incidental findings included pelvic dilation of the right kidney, alopecia of the shoulder and abdominal region, reddish hard nodule on the left uterine horn and vagina contains reddish fluid. At the limited incidence observed, these were not considered to be toxicologically relevant.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment related microscopic findings.
A definitive cause of death could not be established for female no. 67. The nodules in the uterus of animal no. 58 seen at the macroscopic examination represented implantation sites.
There were no microscopic findings in any of the animals suspected of infertility which could explain their lack of reproductive performance.
The remaining recorded microscopic findings were within the range of background pathology encountered in Wistar (Han) rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No signs of parental toxicity were observed.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related effects.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No treatment related effects.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment related effects.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related effects.
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Three pups of the control group and two, two and eight pups of the 100, 300 and 1000 mg/kg groups, respectively were found dead or went missing during the first days of lactation. Missing pups were most likely cannibalized. Seven of the eight dead pups at 1000 mg/kg were all from female no. 80 who had three living and seven dead pups at first litter check. In the absence of any adverse effects seen for any other female in at this dose level, it was not considered to be treatment related. No toxicological relevance was attributed to any of these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
Pale or lean appearance, no milk in the stomach and red spot on the head were incidental clinical signs noted. The nature and limited occurrence of these clinical signs remained within the range considered normal for pups of this age, and were not considered toxicologically relevant.

BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment up to 1000 mg/kg.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings of pups that were found dead included no milk in the stomach and cannibalism of the whole body except the hind legs (noted for three of the seven dead pups from female no. 80). There were no macroscopic findings noted for surviving pups. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

OTHER FINDINGS (OFFSPRING)
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed at any of the dose levels tested.

Effect levels (F1)

Dose descriptor:
NOAEL
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 signs of reproductive toxicity were observed.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

ANALYSIS OF DOSE PREPARATIONS

Samples of Day 6

> Accuracy of preparation:

- No test material was detected in the control group.

- The concentrations analysed in the 100, 300 and 1000 mg/kg bw/day formulations were not in agreement with target concentrations since the mean accuracies were outside the acceptability range of 85-115%; with mean accuracies of 84%, 117% and 130%, respectively.

> Homogeneity:

The 100 and 1000 mg/kg bw/day formulations were not homogeneous since the coefficients of variation were 17% and 42%, respectively.

> Stability:

Analysis of Group 2 and Group 4 formulations after storage yielded a relative difference of 27% and -24%. The relatively large differences are probably caused by an inhomogeneity in the formulations rather than degradation of the test material.

 

Samples of Day 10

> Accuracy of preparation:

- No test material was detected in the control group.

- The concentrations analysed in the 100, 300 and 1000 mg/kg bw/day formulations were in agreement with target concentrations, mean accuracies were between 85% and 115%.

> Homogeneity:

The homogeneity of the 100 and 1000 mg/kg bw/day formulations was still above the criterion of 10%. However, the resulting coefficients of variation were 14% and 11% respectively, were still considered acceptable.

> Stability:

Analysis of 100 and 1000 mg/kg be/day formulations after storage yielded a relative difference of 12% and -9.4%. The formulations are found to be stable when the relative difference is ≤ 10%. Since the relative difference of the 100 mg/kg bw/day formulations of 12% was an increase in concentration, no degradation occurred. The relatively large differences are probably caused by an inhomogeneity in the formulations. The analytical method showed a higher variability at the low concentration level (i.e. a coefficient of variation of 11 and 18% of the procedural recovery samples) which possibly results in a higher variability of the analytical formulation results as well. The formulations are thus considered to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.

Table 1: Reproductive Data Summary

Parameter

Dose Group mg/kg bw/day

Control (0.0)

100

300

1000

Females paired

10

10

10

10

Females mated

10

10

9

10

Females pregnant

10

9

9

10

Non-pregnant females

0

1

0

0

Non-mated females

0

0

1

0

Females with living pups on Day 1

10

9

9

10

Females dead during delivery

0

0

1

0

Mating Index (%)

100

100

90

100

Fertility Index (%)

100

90

90

100

Conception Index (%)

100

90

100

100

Gestation Index (%)

100

100

100

100

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, no signs of parental or reproductive toxicity were observed at dose levels up to 1000 mg/kg bw/day. Based on these findings the NOAEL for parental and reproductive toxicity were determined to be the highest dose level, 1000 mg/kg be/day.
Executive summary:

Toxicity to reproduction was determined in a reproduction/development toxicity screening test, performed under GLP conditions and in line with the standardised guidelines OECD 421 and EPA OPPTS 870.3550. Based on the results of a 10-day dose range finding study, Crl:WI(Han) rats were dosed at 100, 300 and 1000 mg/kg bw/day, formulated in propylene glycol, and administered via oral gavage.

Ten males per dose were exposed for 29 days (2 weeks prior to mating, during mating, and up to termination), and ten females per dose were exposed for 39-54 days (during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation).

The accuracy, homogeneity and stability results were outside the acceptable range during the first formulation analysis. Subsequent analyses revealed the formulations were prepared accurately and were considered stable for at least 6 hours at room temperature, though formulations were not homogeneous. The results were attributable to an interaction between limited sensitivity of the analytical method and the nature of the test material itself and were ultimately accepted. The inhomogeneity of the test material had no negative impact on the study as formulations were stirred constantly during dosing.

No signs of parental toxicity were noted in males or females during exposure or at necropsy up to the maximum dose level tested. Thus the NOAEL for parental toxicity was considered to be 1000 mg/kg bw/day.

Examination of the offspring revealed no signs of treatment related toxicity.

No toxicologically relevant effects on reproductive parameters were noted. There were 10, 9, 9 and 10 pregnant females in the control, 100, 300 and 1000 mg/kg groups, respectively. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. Based on these findings the NOAEL for reproductive toxicity was determined to be the highest dose level, 1000 mg/kg bw/day.