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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 July 2012 to 5 October 2012
Reliability:
1 (reliable without restriction)
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
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid (unspecified)
Details on test material:
- Appearance: Red-brown liquid
- Storage conditions: At room temperature in the dark
- Stability under storage conditions: Stable

Test animals

Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI(Han) (outbred, SPF-Quality).
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: The average weight of the males was 323 g and the average weight of the females was 206 g on Day 1 of the study.
- Housing: Pre-mating, animals were housed in groups of 5 animals/sex/cage in plastic cages (height 18 cm). During mating, females were caged together with males on a one-to-one-basis in plastic cages (height 18 cm). Post-mating, males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in plastic cages (height 18 cm). During lactation, pups were kept with the dam until termination in plastic cages (height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 °C
- Humidity (%): 40 to 70 % relative
- Air changes (per hr): approximately 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark cycle

IN-LIFE DATES: From: 21 August 2012 To: 5 October 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
- Specific gravity: 0.92
- Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for the specific gravity of the test material and vehicle. No correction was made for the purity/composition of the test material. Formulations were placed on a magnetic stirrer during dosing.
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. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The purpose of the analytical phase was to determine the accuracy of preparation, homogeneity and stability of the test material in formulations.
Samples of dose preparations were taken on a single occasion during the treatment phase.
Formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in the vehicle over 6 hours was also determined (highest and lowest concentration).

SAMPLES
Duplicate samples (approximately 500 mg), taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 20 mL. 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 with tetrahydrofuran and analysed.

ANALYTICAL CONDITIONS
Instrument: Alliance Separation Module 2695 (Waters, Milford, MA, USA)
Detector: Refractive Index Detector 2414 (Waters)
Column: 300 x 7.8 mm i.d. PL Gel 50 Å (Waters); 3 columns in series
Column temperature: 40 ± 1 °C
Injection volume: 10 μL
Eluent: Tetrahydrofuran
Flow: 1.0 mL/min
RI detection: Temperature, 30 °C; Sensitivity, 4; Time constant, 1 second

PREPARATION OF SOLUTIONS
Stock and spiking solutions
Stock and spiking solutions of the test material were prepared in tetrahydrofuran at concentrations of 3735 to 11 165 mg/L.

Calibration solutions
Calibration solutions in the concentration range of 400 to 10 000 mg/L were prepared from two stock solutions. The end solution of the calibration solutions was tetrahydrofuran.

Procedural recovery samples
Approximately 500 mg blank vehicle was spiked with the test material at a target concentration of 20 or 250 mg/g. 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.

ELECTRONIC DATA CAPTURE
System control, data acquisition and data processing were performed using the programme Empower version 7.00 (Waters, Milford, MA, USA).
Temperature, relative humidity and/or atmospheric pressure during sample storage and/or performance of the studies was monitored continuously using the programme REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA).

FORMULAS
Response (R)
Peak area test material [units]

Calibration curve
R = (a x Cn) + B
where:
Cn = nominal concentration [mg/L]
a = slope [units × L/mg]
b = intercept [units]

Analysed concentration (Ca)
Ca = ((R - b) / a) x ((V x d) / w [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor

Recovery
(Analysed concentration [mg/g] / Nominal concentration [mg/g]) x 100 [%]

Accuracy
(Analysed concentration [mg/g] / Target concentration [mg/g]) x 100 [%]

Relative difference (relative diff.)
((Ct - C0) / C0) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

SPECIFICATIONS
Preparation of formulations was considered acceptable if the mean accuracy was in the range 90 to 110 % of the target concentration and if the coefficient of variation was ≤ 10 %. Formulations were considered stable if the absolute relative difference between the stored and freshly taken samples was ≤ 10 %.

RESULTS
Calibration curves
A calibration curve was constructed using five concentrations. For each concentration, two responses were acquired. Linear regression analysis was performed using the least squares method with a 1/concentration² weighting factor.
Two responses were excluded from the curve since the back calculated accuracy deviated > 15 % from the nominal concentration. The coefficient of correlation (r) was > 0.99.

Procedural recovery samples
The mean recoveries of the 20 mg/g and 250 mg/g procedural recovery samples were 56 and 104 %, respectively. The mean recovery of the 20 mg/g samples was outside the acceptability range of 90 to 110 %. This was probably caused by interference of corn oil at the retention time of the test material.
Due to the nature of the test material and corn oil as the required vehicle this cannot be avoided. This result is considered to be the best result possible.
It was decided to correct the concentrations measured in the formulation samples for the mean recovery of the corresponding procedural recovery samples. The Group 3 samples were corrected for the average mean recovery of the 20 mg/g and 250 mg/g samples.

Test samples
In the Group 1 formulation, no test material was detected. The concentrations analysed in the formulations of Group 2 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90 and 110 %). For the formulation of Group 3, the mean accuracy was below the target concentration (87 % of target). This mean accuracy was slightly out of the range 90 to 110 %, but still considered acceptable.
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10 %). Analysis of Group 4 formulations after storage yielded a relative difference of ≤ 10 %. Analysis of Group 2 formulations after storage yielded a relative difference of >10 % (12 %). Since this was only slightly above 10 % and, in view of the interference of the vehicle on the test material analysis, this result was accepted as the best possible. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.
Duration of treatment / exposure:
Males were exposed for 29 days: 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42 to 45 days: 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
Once daily, 7 days a week
Details on study schedule:
N/A
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 10-day dose range finding study conducted at the testing laboratory.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Parental animals were observed for mortality / viability at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were conducted for all animals after dosing (at no specific time point, but within a similar time period after dosing for the respective animals). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded.

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
- Time schedule: 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.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OTHER: Observations were carried out for haematology, clotting potential, clinical chemistry and neurobehavioural examinations.
Oestrous cyclicity (parental animals):
The numbers of former implantation sites and corpora lutea were recorded for all paired females.
Sperm parameters (parental animals):
Parameters examined in male parental generation: testis and epididymis weight.
From 5 selected males of the control and high dose group and all males suspected to be infertile, slides of the testes were prepared to examine staging of spermatogenesis.
Litter observations:
Each litter was examined to determine the following, if practically possible:
- Mortality/viability: The numbers of live and dead pups on 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 for all animals.
- Bodyweights: 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):
GROSS PATHOLOGY: Yes
Necropsy of parental animals: All males and 5 selected females/group for full examination, though 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. The numbers of former implantation sites and corpora lutea were recorded for all paired females.
- Sacrifice: All males and the 5 selected females/group were deprived of food overnight (maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food. Animals surviving to scheduled necropsy were deeply anaesthetised using isoflurane and subsequently exsanguinated.

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected and fixed: adrenal glands, aorta, brain (cerebellum, mid-brain, cortex), caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve (if detectable) and Harderian gland), female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, lacrimal gland (exorbital), larynx, liver, lung (infused with formalin), lymph nodes (mandibular, mesenteric), nasopharynx, oesophagus, ovaries, pancreas, peyer's patches (jejunum, ileum) if detectable, pituitary gland, preputial gland, prostate gland, rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid if detectable, tongue, trachea, urinary bladder, uterus, vagina and all gross lesions.

Samples of the following tissues and organs were collected and fixed from all remaining animals and females which failed to deliver: cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, vagina and all gross lesions.

Organ weights: The following organ weights and terminal body weight were recorded from the 5 randomly selected animals/sex/group on the scheduled day of necropsy: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus (including cervix), prostate, seminal vesicles including coagulating glands and thyroid including parathyroid.

The following organ weights and terminal body weight were recorded from all remaining males: epididymides and testes.

All organ and tissue samples were processed, embedded and cut at a thickness of 2 to 4 micrometers and stained with haematoxylin and eosin. From the selected 5 males of the control and high dose group and all males suspected to be infertile or which died before mating, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3 to 4 micrometers, and stained with PAS/haematoxylin.

A peer review on the histopathology data was performed by a second pathologist.
The following slides were examined by a pathologist:
- The preserved organs and tissues of the randomly selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 and the male suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of the male that failed to sire and the female that failed to deliver healthy pups.

* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.
Postmortem examinations (offspring):
SACRIFICE
Pups surviving to planned termination were killed by decapitation on Days 5 to 7 of lactation.

GROSS NECROPSY
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 analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (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 (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.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the 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.
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.
Reproductive indices:
For each group, the following calculations were performed:
- 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:
For each group, the following calculations were performed:
- 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 = (Number of dead pups before planned necropsy / Number of live pups at first litter check) x 100
- Viability index = (Number of live pups before planned necropsy / Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
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:
not examined

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 CLINICAL SIGNS
No mortality occurred during the study period.
No clinical signs of toxicity were noted during the observation period.

BODY WEIGHTS
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.

HAEMATOLOGY AND CLOTTING FACTORS
Any changes were considered to be of no toxicological relevance as the changes were slight, they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.

CLINICAL BIOCHEMISTRY
Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals. The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

MACROSCOPIC EXAMINATION
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

ORGAN WEIGHTS
No toxicologically relevant organ weight changes were noted up to 1000 mg/kg for both sexes.

MICROSCOPIC EXAMINATION
There were no microscopic findings in any of the animals suspected of infertility which could explain their lack of reproductive performance. The spermatogenic staging profiles were normal for all animals assessed. There were no treatment related microscopic findings.

REPRODUCTION DATA
No toxicologically relevant effects on reproductive parameters were noted.
Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Detection of mating was not confirmed for animal no. 65 (Group 3) 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.

DEVELOPMENTAL DATA
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.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No parental toxicity was observed.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effects on reproductive parameters were noted.
Remarks on result:
other: Generation: Reproductive Parameters (migrated information)

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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Two pups of the control group were found dead during the first days of lactation. This remained within the range considered normal for pups of this age and as it concerned control pups only it was not treatment related.

CLINICAL SIGNS (OFFSPRING)
One pup of the control group showed a pale appearance on Days 1 and 2 of lactation. At macroscopic examination, two pups of one litter at 1000 mg/kg showed absence of milk in the stomach. The nature and incidence of these observations remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT (OFFSPRING)
Body weights of pups were considered to have been unaffected by treatment. The statistically significantly increased values noted at 300 mg/kg were not considered toxicologically relevant as the values were within normal, no dose response relationship was noted, and these increased weights were due to two litters with smaller litter sizes.

Effect levels (F1)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Precoital Time (F0 Generation Postcoitum)

Day of the Pairing Period

Dose group (mg/kg)

Control

100

300

100

Number of Females Mated

1

2

3

4

5

3

1

4

2

-

3

1

4

1

1

6

-

2

1

1

4

2

2

2

-

Median Precoital Time

Mean Precoital Time

N

3

2.5

10

3

2.6

10

1

2.1

10

2

2.2

10

 

Table 2: Summary of Reproduction Data

 

Dose group (mg/kg)

Control

100

300

100

Females paired

Females mated

Pregnant females

Non-pregnant females

Females with living pups on Day 1

10

10

10

0

10

10

10

9

1

9

10

10

10

0

10

10

10

10

0

10

Mating Index (%)

Fertility Index (%)

Conception Index (%)

Gestation Index (%)

100.0

100.0

100.0

100.0

100.0

90.0

90.0

100.0

100.0

100.0

100.0

100.0

100.0

100.0

100.0

100.0

 

Table 3: Summary of the Number of Corpora Lutea and Implantation Sites at Necropsy

 

 

Dose group (mg/kg)

Control

100

300

100

Corpora lutea

Mean

SD

N

15.3

1.6

10

14.2

2.1

9

14.4

3.1

10

15.7

1.6

10

Implantations

Mean

SD

N

13.3

2.0

10

12.9

2.1

9

12.5

2.5

10

13.1

2.6

10

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the NOAEL was determined to be 1000 mg/kg bw/day for reproductive parameters.
Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guidelines OECD 422 and EPA OPPTS 870.3650 under GLP conditions.

Four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test material at 0, 100, 300 and 1000 mg/kg/day.

Males were exposed for 29 days: 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42 to 45 days: 2 weeks prior to mating, during mating, during post-coitum and during at least 4 days of lactation.

Animals were evaluated for mortality / viability, clinical signs, functional observations and locomotor activity, bodyweight and food consumption, clinical pathology, macroscopy at termination, organ weights and histopathology on a selection of tissues and reproduction / developmental parameters.

No parental toxicity was observed up to 1000 mg/kg.

Under the conditions of this study, the NOAEL was determined to be 1000 mg/kg bw/day for reproductive parameters.