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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:
13 October 2010 - 24 November 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)
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name: Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
Physical state: liquid
Appearance: brown liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Amine C8
- Physical state: dark liquid
- Storage condition of test material: at room temperature, 10 to 27 °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) a minimum of 13 weeks old at initiation of cohabitation; records of dates of birth for animals used in this study will be retained in the Calvert archives
- Weight at study initiation: (P) Males: > or = 300 g; Females: > or = 200 g at initiation of cohabitation
- Fasting period before study: no data
- Housing: Upon arrival and until randomization, males and females are group-housed, sexes separate. Following randomization and until cohabitation, males and females will be individually housed. During cohabitation, one female will be placed with a male breeder from the same group. Following cohabitation, males and females will be housed individually. No later than gestation Day 17, mated female animals are plaec in totes with bedding. Animals will be housed in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals". The room in which the animals are kept is documented in the study records. No other species are kept in the same room
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): All animals have access to Harlan Teklad Rodent Diet (certified) or equivalent ad libitum, unless otherwise specified. No contaminants are known to be present in the certified diet at levels that would be expected to interfere with the results of this study. Analysis of the diet was limited to that performed by the manufacturer, records of which will be maintained in the Calvert archives.
- Water (e.g. ad libitum): Water will be available ad libitum, to each animal via an automatic watering device. The water routinely analyzed for contaminants as per Calvert SOP's. No contaminants are known to be present in the water at levels that would be expected to interfere with the results of this study. Results of the water analysis will be maintained in the Calvert archives.
- Acclimation period: Study animals will be acclimated to their housing for a minimum of 7 days prior to their first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26°C
- Humidity (%): 30 to 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark, except when room lights are turned on during the dark cycle to accomodate blood sampling or other study procedures

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test article and vehicle control preparations are prepared weekly or additionally as needed by diluting the test article in vehicle (w/v) to reach the proper concentrations.

Dose formulation samples:
On the first day of dosing, at the beginning of cohabitation and at the last day of dosing, duplicate 1-mL samples are obtained from top, middle, and bottom of each formulation, including the vehicle control, to determine the concentration and homogeneity of the test article in the vehicle. These samples are stored at room temperature, approximately 10 to 30°C.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Details on mating procedure:
Animals are mated by placing one male and one female from the same dose group overnight in a breeding cage until evidence of copulation is noted, after which the male animal is separated. Animals remain in cohabitation for a maximum of three weeks. During this time, the study Director may elect to move certain females with other males from the same dose group, in an attempt to expedite mating. This procedure is documented in the study raw data records.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Males: The vehicle control or test article formulations are administered once daily for a minimum of four weeks (starting two weeks prior to cohabitation). Treatment continues during the same group cohabitation period and until the day before scheduled for euthanasia. Each animal received a mL/kg dose based upon its most recent body weight.
Females: The test/vehicle control or test article formulations are administered once daily for a minimum of 15 days prior to cohabitation, during cohabitation and from presumed gestation days 0 through day 19 of gestation.
Procedure: Each animal receives a mL/kg dose based upon its most recent body weight.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 other: mg/kg
Remarks:
Basis: nominal conc. control vehicle
Dose / conc.:
1 000 other: mg/kg
Remarks:
Basis: actual ingested gavage
No. of animals per sex per dose:
10
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 studyes
- Rationale for animal assignment (if not random): at random

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality: twice daily; once prior to scheduled sacrifice
- Cage side observations: each animal is observed for evidence of death or impending death (as per Calvert SOP VET-14)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: throughout the treatment phase, a minimum of twice daily, prior to dose administration and a minimum of once following dosing. On non-dosing days, a minimum of once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: males: once weekly prior to initiation of cohabitation, during cohabitation and at terminal sacrifice. A final body weight is also obtained for all males sacrificed moribund; females: once weekly prior to initiation of cohabitation on gestation days 0, 4, 7, 10, 14, 17 and 20 (day 23 and 26 if required), and on Day 0 and 4 of lactation. A final body weight is also obtained for all females showing signs of premature delivery or sacrificed moribund.

FOOD CONSUMPTION:
Males and females: frequency: full feeder weights and/or feeder weigh backs are recorded once weekly prior to cohabitation, on gestation days 0-4, 4-7, 7-10, 10-14, 14-17, 17-20 (20-23 and 23-26 if required), and on Day 0 and 4 of lactation. During cohabitation food consumption is not recorded

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

Oestrous cyclicity (parental animals):
Estrous cycle evaluation is performed daily for 2 weeks prior to cohabitation and daily during the treatment and cohabitation periods. Day 0 of gestation is determined by evidence of copulation which is determined by the examination of vaginal smears made daily to determine if sperm are present in a smear of vaginal contents or by the presence of a copulatory plug in situ. Examination of vaginal smears is performed at approximately the same time each day (early morning) throughout the cohabitation period.
Postmortem examinations (parental animals):
SACRIFICE
a) Method of euthanasia:
All adult animals are sacrificed by CO2 asphyxiation. All male animals are euthanized 2 weeks post-mating and all females are euthanized on Day 4 of lactation. All pups are euthanized by an intrathoracic injection of a barbiturate overdose.
b) Unscheduled deaths of males and females:
For all males and females sacrificed moribund or found dead prior to scheduled sacrifice, a complete gross necropsy is performed. The necropsy includes 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, seminal vesicles and prostate are retained in 10% neutral buffered formalin for possible histopathological evaluation.
Additionally, for male rats, the testes and epididymides are 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 are recorded. Where applicable, the number of viable and non-viable fetuses are also recorded. All fetuses are discarded.
c) Terminal sacrifice of males:
all surviving male animals are euthanized 2 weeks post-mating and a macroscopic examination are performed. The necropsy includes the examination of the external body surface, all orifices and the cranial, thoracic and abdominal cavities and their contents.
d) Terminal sacrifice of females:
All surviving females are euthanized on Day 4 of lactation and a macroscopic examination is performed. The necropsy includes the examination of the external body surface, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The total number of implantation scars are also determined.

GROSS NECROPSY
A complete gross necropsy is performed by Calvert personnel on all adult animals that are sacrificed or found dead during the study. The necropsy includes examination of:
- the external body surface
- all orifices
- the cranial, thoracic and abdonimal cavities and their contents
All abnormalities are described completely and recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
For all male animals at scheduled sacrifice at 14 days post cohabitation, the following organs are weighed before fixation, after dissection of excess of fat and other excess tissues. Organ weights are not recorded for animals found dead or sacrificed moribund: epididymides, testes. Organ to body weight ratios are calculated (using the final body weight obtained prior to necropsy), as well as organ to brain weight ratios.
Tissue collection and preservation: all tissues for all adult animals are examined. For all animals necropsied, the tissues are preserved in 10% neutral buffered formalin (except for the epididymides and testes that will be retained in modified Davidson's fixative for optimum fixation). Tissues collected: ovaries, uterus, cervix, vagina, testes, epididymides, prostate, seminal vescicles. Special emphasis on stages of spermatogenesis and histopathology of the interstitial testicular structure are given.
Postmortem examinations (offspring):
SACRIFICE
All pups are euthanized by an intrathoracic injection of a barbiturate overdose on Day 4 of lactation. All neonates are sexed, weighed, examined as soon as possible after delivery for litter seize, still births, live births and any gross anomalies. Neonates are not retained. All neonate malformations are photographed.

Statistics:
Statistical analysis will be performed on in-life and necropsy data when 3 or more animals are present in 2 or more dose groups. Statistical analysis is not performed if N<3 animals per group. For in-life parameters, the homogeneity of the data is determined by Bartlett's Test. If the data is homogeneous, a one-way analysis of variance is performed to assess statistical significance. If statistically significant differences between the meand are found, Dunnett's test is used to determine the degree of significance from the control means (p<0.05 and p<0.01). If the data is non-homogeneous, the Kruskal-Wallis non-parametric analysis is performed to assess statistical significance. If statistically significant differences between the means are fround (p<0.05, p<0.01), the Mann-Whitney U-Test is used to determine the degree of significance from the control means (p<0.05 and p<0.01). If only 2 dose groups are present for evaluation, the Mann-Whitney U-Test is used to assess statistical significance between the 2 groups. For necropsy organ weight data, the evaluation of the equality of means is made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, Dunnett's test is used to determine the degree of significance from the control means (p<0.05 and p<0.01). If only 2 dose groups are present for evaluation, the Mann-Whitney U-test is used to assess statistical significance between the 2 groups. For necropsy organ weight data, the evaluation of the equality of means is made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, Dunnett's test is used to determine the degree of significance from the control means (p<0.05 and p<0.01).
Reproductive indices:
Pre-coital interval (in days): sum of days until successful copulation/number of presumed pregnant animals
Copulation index (%): (number of presumed pregnant animals/number of paired animals) x 100
Fertility index (%): (number of pregnant animals/number of presumed pregnant animals) x 100
Preimplantation loss (%): (no. of corpora lutea - number of implantations/number of corpora lutea) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female animal was found dead on lactation day 2. All other females survived until scheduled sacrifice.
All male animals survived until scheduled sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant changes in bw or bw gain (in females and males)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No biologically relevant effects on food consumption during study (in females and males)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No statistically significant changes in absolute or relative testes and epididymides weights.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No microscopic toxic effects observed at 1000 mg/kg/dose (males and females)
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
no data
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
no data
Reproductive performance:
no effects observed
Description (incidence and severity):
In Life: One animal was non-gravid, another female did not mate. All males appeared normal.
Post mortem: no definitive effects on percent gravidity. No effects on pre-coital interval, copulation index and duration of gestation period were noted.

Details on results (P0)

no statistically significant findings in the number of gravid animals, corpora lutea/dam, total implantations and pre-implantation loss.
no treatment-related differences in litter viability parameters.
no statistically significant changes in fetal sex ratios.

Effect levels (P0)

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 adverse effects observed

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Incidence of neonates born alive/found dead, stillborn or missing between lactation days 0-4 was comparable among study groups.
One female (1000 mg/kg) was found dead on lactation day 2, and all neonates were stillborn.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
No treatment-related differences in litter viability parameters.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the neonates were statistically significantly increased. The biological significance of this finding is unknown.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
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 malformations were observed for neonates.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No statistically significant changes in fetal sex ratios.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No treatment-related differences in litter viability parameters.

Neotnate Observation
Incidence of neonates born alive/found dead, stillborn or missing between lactation days 0-4 was comparable among study groups.
One female (1000 mg/kg) was found dead on lactation day 2, and all neonates were stillborn.
No treatment-related malformations were observed for neonates.
No statistically significant changes in fetal sex ratios.

BODY WEIGHT (OFFSPRING)
Neonate bw were statistically significantly increased. the biological significance of this finding is unknown.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a screening reproductive / development toxicity test (performed according to OECD guideline 421), rats were exposed to 1000 mg test substance/kg bw/d orally (via gavage). No adverse effects were observed. A NOAEL of 1000 mg/kg bw/d was derived. The test substance is not to classified as reproductive toxicant according to the criterial laid down in the CLP Regulation.