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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Oct 2010 - 6 Dec 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: liquid
- Storage condition of test material: Room temperature
Specific details on test material used for the study:
- Name of test material (as cited in study report): Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)-
- Physical state: clear colorless liquid
- Lot/batch No.: OF502
- Storage condition of test material: at room temp (10-24.6°C)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: (P) x a minimum of 13 weeks at initiation of cohabitation
- Weight at study initiation: (P) Males: >/=300 g; Females: >/=200 g at initiation of cohabitation
- Fasting period before study:
- Housing: Upon arrival and until randomization, males and females were group-housed, sexes separate. Following randomization and until cohabitation, males and females were individually housed. During cohabitation, one female was placed with a male breeder from the same group. Following cohabitation, males and females were housed individually.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): All animals had access to Harlan Teklad Rodent Diet (certified) or equivalent ad libitum.
- Water (e.g. ad libitum): Water was available ad libitum via an automatic watering device.
- Acclimation period: Study animals were acclimated to their housing for a minimum of 7 days prior to the first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark, except when room lights were turned on during the dark cycle to accommodate blood sampling or other study procedures.

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
dermal
Vehicle:
other: deionized water
Details on exposure:
TEST SITE
- Area of exposure: 4cm x 4cm
- % coverage:
- Type of wrap if used:
- Time intervals for shavings or clipplings: The pelage covering the interscapular and dorsal thoracic regions of the body of the study animals were clipped at least 48 hours prior to the first topical dose and additionally as needed.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): In case of excessive test substance buildup the application site was cleansed after consultation with the sponsor.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 5 ml/kg (concentrations of 20, 50 and 80 mg/ml) of the test substance was distributed with a syringe as evenly as possible over the exposure area of the intact skin of each animal.
- Constant volume or concentration used: yes

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 ml/kg

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes; During the dosing period the rats were fitted with BiteNot collars 8 hours a day to prevent the animal from licking the test substance.
Details on mating procedure:
- M/F ratio per cage: 1 male to 1 female from the same group
- Length of cohabitation: Males and females remained together until evidence of copulation was noted or for a maximum of three weeks.
- Proof of pregnancy: Day 0 of gestation was determined by evidence of copulation which was determined by the examination of vaginal smears made daily to determine if sperm were present in a smear of vaginal contents or by the presence of a copulatory plug in situ. Examinations of vaginal smears were performed at approximately the same time each day (early morning) through the cohabitation period.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: After three weeks of cohabitation, the Study director was able to elect to move certain females with other males from the same dose group, in an attempt to expedite mating.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): Following cohabitation, the females were housed individually.
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first day of dosing, at the beginning of cohabitation and at the last day of dosing, duplicate 1-ml samples were obtained from top, middle and bottom of each formulation, including the vehicle control, to determine the concentration and homogeneity of the test substance in vehicle. In addition, samples were tested for stability. These samples were stored at room temperature, approximately 10 to 30 degrees C.
Duration of treatment / exposure:
The males were treated once daily for a minimum of four weeks (starting two weeks prior to cohabitation). Treatment continued during the same-group cohabitation period and until the day before sacrifice (Day 28/euthanasia Day 29). Females were treated once daily for a minimum of 15 days prior to cohabitation, during cohabitation and from presumed Gestation Days 0 through Day 19 of gestation.
Frequency of treatment:
once daily
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: [...] weeks
Doses / concentrationsopen allclose all
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10/sex/group (80 animals)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based upon previously conducted toxicity studies. A dermal dose range finding study was performed by Calvert labs (report 0325RH11.001, dated March 21, 2011). Test article was dosed by topical application to 80 SD rats (40 males, 40 females) approximately 11 weeks old at initiation. Four dose levels used: control, 100 mg/kg, 250 mg/kg, 400 mg/kg. Due to necrosis and thickening of the skin at the test article application site, dosing for group 3 and 4 was discontinued on day 2 and animals were euthanised. On day 4, similar effects were observed in the dosing group 2. Due to these severe skin effects observed in the 100-400 mg/kg/d dose groups, 24 additional animals were added to the study in order to evaluate dermal effects at lower dose levels: 8 animals (4 male/4 female) were divided over 3 dose groups: 10 mg/kg, 25 mg/kg and 75 mg/kg. The dose was applied once daily for 7 days. Dose administration was discontinued for the 75 mg/kg dose group on day 3 (necrosis). Surviving animals (dose group 10 and 25 mg/kg) were sacrificed on day 7.
Gross necropsy was performed (examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents; no tissues were retained).
Clinical signs in animals dosed at 25 mg/kg were limited to very slight to well defined erythema and fissuring at the dosing site of some male and female animals. The NOAEL was considered to be 10 mg/kg.

- Rationale for animal assignment: Using a random number generator, study animals were assigned a random number, sorted based upon the random number assignments, and then assigned to groups. Using SYSTAT version 9.0.1, an Analysis of Variance followed by Dunnett's test was performed to confirm that there were no significant differences in mean body weights between the study groups. If the group assignments showed statistical significane, animals were allowed to be reassigned within the order to improve the similarity of group mean body weights.

Positive control:
N/A

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was observed twice daily (a.m. and p.m.), once prior to scheduled sacrifice. Clinical observations were made throughout the treatment phase, a minimum of twice daily, prior to dose administration and a minimum of once following dosing. On non-dosing days, observations were made a minimum of once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed once weekly prior to initiation of cohabitation, during cohabitation and at terminal sacrifice. A final body weight was also obtained for all males sacrificed moribund. Females were weighed once weekly prior to initiation of cohabitation, on Gestation Days 0, 4, 7, 14 and 20, and on Day 0 and 4 of lactation. A final body weight was also obtained for all females showing signs of premature delivery or sacrificed moribund.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Full feeder weights and/or feeder weight backs were recorded once weekly, except during mating.

Oestrous cyclicity (parental animals):
Estrous cycle evaluation was performed daily for 2 weeks prior to treatment initiation and daily during the treatment and cohabitation periods.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed by CO2 asphyxiation 2 weeks post-mating.
- Maternal animals: All surviving animals were sacrificed by CO2 asphyxiation on Day 4 of lactation.

GROSS NECROPSY
For all males and females sacrificed moribund or found dead prior to scheduled sacrifice, a complete gross necropsy was performed. The necropsy included the examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The ear tag, all gross lesions, ovaries, uterus, cervix, vagina and prostate were retained in 10% neutral buffered formalin for possible histopathological evaluation. Additionally, for male rats, the testes and epididymides were retained in modified Davidson's fixative for possible histopathological evaluation. Where applicable, the total number of implantation sites and the total number of corpora lutea for each ovary were recorded. Where applicable, the number of viable and non-viable fetuses were also recorded. All fetuses were discarded.
For the terminally sacrificed males (2 weeks post-mating), necropsy included the examination of the external body surface, all orifices and the cranial, thoracic and abdominal cavities and their contents.
For the terminally sacrificed females (Day 4 of lactation), necropsy included the examination of the external body surface, all orifices and the cranial, thoracic and abdominal cavities and their contents. The total number of corpora lutea were determined for each ovary, and the total number of implantation sites, viable and non-viable fetuses, and early or late resorptions were also determined. For presumed non-gravid females, the uterus was stained with 10% ammonium sulfide to assess the presence of implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
The epididymides and testes were weighed before fixation, after dissection of excess fat and other excess tissues, for all males surviving until the scheduled sacrificed day. Organ to body weight ratios were calculated (using the final body weight obtained prior to necropsy), as well as organ to brain weight ratios.
All tissues for all animals were examined. For all animals necropsied, the following tissues were preserved in 10% neutral buffered formalin (except for the epididymides and testes that were retained in modified Davidson's fixative for optimum fixation): ovaries, uterus, cervix, vagina, testes, epididymides, prostate, seminal vesicles, and gross findings. Special emphasis on stages of spermatogenesis and histopathology of the interstitial testicular structure were give.
Tissues for evaluation were processed to paraffin blocks and prepared to slides. Slides were stained with hematoylin and eosin. Occasionally, other stains may have been required to aid in the diagnosis of lesions; these were documented when used.
Slides were prepared for all tissues for all rats in the vehicle and high dose groups and for all animals that died early. If test substance related lesions were noted, additional slides were prepared on those tissues from the low and mid dose groups.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were euthanized by an intraperitoneal injection of a barbiturate overdose on day 4 of lactation.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
Statistical analysis was performed on In-Life, Clinical Pathology, and Necropsy data when 3 or more animals were present in 2 or more dose groups. Statistical analyses were not performed if N<3 animals per group. For In-Life and Clinical Pathology parameters, the homogeneity of the data was determines by Bartlett¿s Test. If the data was homogeneous, a one-way analysis of variance was performed to assess statistical significance. If statistically significant differences between the means were found, Dunnett¿s test was used to determine the degree of significance from the control means (p<0.05 and p<0.01). If the data was non-homogeneous, the Kruskal-Wallis non-parametric analysis was performed to assess statistical significance. If statistically significant differences between the means were found (p<0.05, p<0.01), the Mann-Whitney U-Test was used to determine the degree of significance from the control means (p<0.05 and p<0.01). If only 2 dose groups were present for evaluation, the Mann-Whitney U-test was used to assess statistical significance between the 2 groups. For necropsy organ weight data, the evaluation of the equality of means were made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, Dunnett¿s test was used to determine the degree of significance from the control means (p<0.05 and p<0.01).
Reproductive indices:
1. Pre-coital interval (in days): sum of days until successful copulation/# of presumed pregnant animals
2. Copulation index (%): # of presumed pregnant animals/# of paired animals x 100
3. Fertility index (%): # of pregnant animals/# of presumed pregnant animals x 100
4. Preimplantation loss (%): # of corpora lutea-# of implantations/# of corpora lutea x 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

No effects were observed, except some local irritating effects at the site of administration at 30 mg/kg bw.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: male and female reproductive performance
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parental clinical signs

Results: F1 generation

Details on results (F1)

No effects were observed.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no treatment-related effects on pregnancy status; gestation, viability and related parameters; fetal weights and fetal morphological observations. There were no test article related changes in fetal sex ratios.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No systemic effects were observed at the highest dose administrated. Therefore a NOAEL of 30 mg/kg bw/d is derived for male and female reproductive performance. The NOAEL for parental clinical signs was considered to be 10 mg/kg bw/d. There were no treatment-related effects on pregnancy status; gestation, viability and related parameters; fetal weights and fetal morphological observations.