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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 July 2014 to 11 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,3-Bis(aminomethyl)cyclohexane
- Physical state: liquid
- Storage condition of test material: Room temperature, in the dark. The container must be kept tightly sealed with nitrogen when not in use.

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK).
- Age at study initiation: Approximately 71 days old
- Weight at study initiation: 233 to 286 g.
- Housing: for mating, the females were housed 1:1 with identified stock males, on the day of positive evidence of mating (Day0) one female per cage
steel mesh lid;
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: At least five days before commencement of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19-23ºC
- Humidity (%):40-70%
- Photoperiod (hrs dark / hrs light):12 hours light : 12 hours dark

IN-LIFE DATES: From: 2 July 2014 To:28 July to 31 July 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was weighed out to give the required amount in a suitable container. Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the test material.The liquid was then stirred using a magnetic stirrer until evenly mixed. The solution was made up to final volume with purified water and mixed using a magnetic stirrer until homogenous. The remaining formulations were made in ascending order of concentration.

VEHICLE

- Concentration in vehicle: 0, 6, 20 and 60 mg/ml
- Amount of vehicle (if gavage):5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical method GC-FID to check achieved concentration of samples of the first and last formulations prepared for administration and stability from the last formulation. Stability was assessed after six hours at ambient storage.

Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From Day 6 to Day 19 of gestation
Frequency of treatment:
Once daily
Duration of test:
20 days (from Day 0 to Day 20 of gestation)
No. of animals per sex per dose:
20 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:The doses used in this study (0, 30, 100 and 300 mg/kg/day) were chosen based on the findings from a Repeated toxicity and
Reproductive/Developmental Toxicity test with 1,3-Cyclohexanedimethanamine in Crl:CD(SD) Rats (Sponsor study Number: B041798).
- Rationale for animal assignment (if not random):To group and cage position in the sequence of mating. Females mating on any one day were evenly
distributed amongst the groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: start and end of each working day
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily 1 to 2 hours after completion of dosing and as late as possible in the working day.
detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health

BODY WEIGHT: Yes
- Time schedule for examinations:weight of each adult was recorded on Days 0, 3, 6 to 20 after mating

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Uterus Gravid uterine weight (including cervix and ovaries)
Full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:Fetuses (live and dead).
Fetal examinations:
- External examinations: Yes: all per litter, sex of each fetus was recorded
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
Statistics:
The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
Bartlett's test for variance homogeneity
Williams’ test for parametric monotonic trend test
Dunnett's test for non monotonic trend test
Kruskal-Wallis’ test for non parametric analysis if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.
Wilcoxon rank sum tests for significant (p<0.05) inter group comparisons
H1 approximate test for monotonicity of dose-response
Shirley's test, a non parametric monotonic trend test, for comparison of the means
Steel's test if the H1 approximate test was significant, suggesting that the dose-response was not monotone

Litter size and survival indices and fetal, placental and litter weight and gravid uterine weight data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests.

Pre/post implantation loss and sex ratio were analysed by a random test (Lipsitz).

For resorptions, each treated group was compared to control by exact Wilcoxon rank sum test.
Indices:
Prenatal losses are separated into pre- and post-implantation phases.

Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations)/ Number of corpora lutea x 100

Post-implantation loss (%) = (Number of implantations –Number of live fetuses)/Number of implantations x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The oral administration of 1,3-BAC at 300 mg/kg/day was not tolerated by pregnant females. Three females were sacrificed for welfare reason as they were considered in general poor clinical condition, with signs including underactivity, gasping, piloerection, salivation and rales (noisy breathing), macroscopic examination revealed abnormal contents (gas) in the colon, ileum, jejunum, and stomach, and also revealed partially blocked nasal turbinates. Females receiving 300 mg/kg/day showed slight bodyweight loss between Days 6 and 7 of gestation and rales was also observed in six females given 300 mg/kg/day and in three females receiving 100 mg/kg/day.
For females which received 1,3-BAC at 30 or 100 mg/kg/day, there was no adverse effect of treatment on bodyweight, bodyweight change, adjusted bodyweight or food consumption.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Placental weight was slightly reduced, when compare with the controls, in females given 300 mg/kg/day, there was, however, no effect on litter weight, or fetal weight.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Placental weight was slightly reduced, when compare with the controls, in females given 300 mg/kg/day, there was, however, no effect on litter weight, or fetal weight.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was considered to be no effect of treatment on litter parameters.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was considered to be no effect of treatment on litter parameters.
Changes in litter size and weights:
no effects observed
Skeletal malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
All females receiving 1,3-BAC at 300 mg/kg/day were found to be pregnant at macroscopic examination on Day 20 after mating. Litter data, as assessed by the mean numbers of corpora lutea, implantations, resorptions, live fetuses, sex ratio, and the extent of pre- and post-implantation loss were considered unaffected by treatment. Although the mean sex ratio at 300mg/kg/d was statistically lower than in Controls, the magnitude of difference from the ideal value of 50% was similar to controls and there was no effect of treatment on embryo-fetal survival or the morphology of the reproductive organs. Placental weight was slightly reduced, however there was no effect on litter or fetal weights and there were no treatment related fetal pathology examinations.
Thus, the data showed that there is no evidence to indicate that 1,3-BAC is a developmental toxicant at any of the doses tested, up to 300mg/kg/day.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Formulation analysis: the stability was confirmed for 1,3-BAC in water formulations and the mean concentrations of 1,3-BAC in test formulations analysed for the study were within ±8% of nominal concentrations, confirming accurate formulation.

Applicant's summary and conclusion

Conclusions:
Treatment with 1,3-BAC at 30 or 100 mg/kg/day was well tolerated, with no effects of treatment on embryo-fetal growth, survival or development. Treatment with 1,3-BAC at 300 mg/kg/day exceeded the maximum tolerated dose for pregnant females but was not associated with adverse effects on embryo-fetal development. The maternal and fetal no observed adverse effect level (NOAEL) was therefore considered to be 100 mg/kg/day.