Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.4.-9.7.2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Dicyclohexylamine
- Substance type: monoconstituent organic substance
- Physical state: liquid
- Analytical purity: 99.63%
- Impurities (identity and concentrations): organic impurities 0.32%, water <0.05%
- Composition of test material, percentage of components: 99.63% main constituent
- Lot/batch No.: 89037152
- Expiration date of the lot/batch: 03/2015
- Stability under test conditions: stable
- Storage condition of test material: at laboratory temperature

Test animals

Species:
rat
Strain:
other: Wistar CRL
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Únětice, Czech Republic
- Age at study initiation: 11 weeks
- Weight at study initiation: approx. 230-260 g
- Housing: SPF special animal room, plastic cages containing sterilised clean shavings of soft wood, before mating 2 rats of the same sex in one cage, during mating period – one male and two females in one cage. Pregnant females were placed individually.
- Diet (e.g. ad libitum): Complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN)
- Water (e.g. ad libitum): Free access to drinking water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Relative humidity (%): 30-70%
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle

STUDY TIME SCHEDULE
Mating: 22. 4. 2014 – 01.5.2014
Start of administration: 28. 4. 2014
End of administration: 20. 5. 2014
Clinical observation: 28.4. – 20. 5. 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was weighted into glass beaker and the beaker was replenished by olive oil. The application form was placed into ultranonic bath for a 5min. After this the application form (suspension) was stirred by magnetic stirrer for 15 min at 800 rpm.

VEHICLE
- Justification for use and choice of vehicle: test sbstance insoluble in water
- Amount of vehicle (if gavage): 1 mL of suspension per 100 g of body weight
- Lot/batch no. (if required): 5464101

STABILITY AND HOMOGENITY OF THE PREPARATION: From the results of analyses (homogeneity and stability) follows that the solution of the test substance in vehicle prepared at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 120 minutes starting with preparation of the application form.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1M/2F
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
:
Duration of treatment / exposure:
from implantation (the 5th day after fertilization) to one day prior to the day of scheduled euthanasia (the 19th day after fertilization)
Frequency of treatment:
daily
Duration of test:
15 days
No. of animals per sex per dose:
24 pregnant females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The evaluation of data about toxicity of Dicyclohexylamine was performed. This evaluation of toxicity information will serve for the determination of dose levels for Repeated Dose 90-day Oral Toxicity Study and Prenatal Developmental Toxicity Study. The data used for toxicity evaluation of above mentioned substance originated from three sources:
- Original literature data obtained from internet
- Data from toxicological databases – free and commercial
- (Q)SAR Evaluation
According to information mentioned above, especially based on the results of 28-day repeated dose study and reproduction toxicity screening test, the following doses – 0, 40, 80 and 160 mg/kg bw/day were chosen for Prenatal Developmental Toxicity Study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during administration period

BODY WEIGHT: Yes
- Time schedule for examinations: on the 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: 20th day
- Organs examined: uterus

MACROSCOPICAL EXAMINATION: Yes
- external surface of the body, all orifices and the cranial, thoracic and abdominal cavities
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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: included in Soft tissue examinations and Skeletal examinations
Statistics:
For statistical evaluation the software Statgraphic ® Centurion (version XV, USA) was used. The data from control group were compared with data from treated groups. The results statistically significant on probability level 0.05 were indicated.
The parametric tests were used for statistical evaluation of:
- body weight of females
- mean weight of fetuses (males, females, both sex)
- biometry of uterus (absolute and relative weight )
The non-parametric tests were used for statistical evaluation of following parameters:
- number of corpora lutea, number of implantations, number of resorptions
- number of live fetuses (males, females, both sex)
The categorical data (external and internal alteration) was summarised in the form of a summary tables but test of independence between row and column classifications was not implemented by the reason of low incidence of these findings at the treated group against the control.
Indices:
preimplantation loss - IUDE (Intra Uterine Death Early): (corpora lutea – implantations)/corpora lutea*100
postimplantation loss - IUDL (Intra Uterine Death Late): resorptions/implantations*100
Historical control data:
Not available

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Strong toxic effect of the test substance on maternal animals was observed. The majority of the endpoints of maternal toxicity (includes mortality, body weight changes, health clinical changes, changed food consumption, changes in uterus weight, pathological findings, changed reproduction parameters) were found out in treated females and they are described below.
The statistically significantly decreased body weight of treated females (related to lower body increment) and seriously changed health condition of females (piloerection, convulsion, tremble, salivation, gibbous posture, apathy, vocalism) at the middle and at the highest dose levels was detected. The number of females with pathological findings was increased in the highest dose. Other finding related with treatment was enlargement of the adrenal gland at the highest dose (releasing hormones in response to stress). The biometry of uterus revealed decreased absolute and slightly decreased relative weight of uterus (except the relative weight of uterus at the middle dose).
Reproduction parameters – number of live foetuses, early and late intra uterine death were evaluated on the basis of examination of uterus content. During examination of reproductive parameters the following changes were recorded - the decreased number of implantations, corpora lutea at the middle and highest dose level and increased number of resorptions at the lowest dose level. The preimplantation loss was increased at the highest dose level and intra uterine death after implantation was increased at the lowest dose level.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
< 40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The low incidence of variations and malformation (in foetuses of treated dams compared to control dam) can not be considered as teratogenic effect of the test substance on early prenatal development of organism in uterus. The causes of their occurrence are questionable, it could be influenced by many factors, one of them is genetic background.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect (the highest dose)

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

For detailed information on results - see attached document.

Applicant's summary and conclusion

Conclusions:
The toxicity in maternal animals administered by test substance was observed.
The delayed development of foetuses (delayed ossification of cranial, sternum skeleton and sacral vertebrae) was detected at all groups (including control group) but is not induced by the test substance treatment and can be connected with low individual body weight of foetuses. The lowered body weight of foetuses is noticeable especially in highest dose group.
The structural alterations in development were found out in all groups. One variation (wavy ribs) was detected in the litters of lowest dose level and also in control group. The other variations (sternum – misaligned sternebrae and increased number of floating ribs) were sporadically detected in treated groups and not in control. The more serious structural alteration - malformation (fused ribs – cartilaginous fusion) occured only in one foetus from the middle dose level.
The low incidence of variations and malformation (in foetuses of treated dams compared to control dam) can not be considered as teratogenic effect of the test substance on early prenatal development of organism in uterus. The causes of their occurrence are questionable, it could be influenced by many factors, one of them is genetic background.
Therefore the NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT is higher than 160 mg/kg/day.

The toxicity in maternal animals administered by test substance was observed in all treated groups.
The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES is less than 40 mg/kg/day. This NOAEL value is based on the death of two females, the statistically significant decreased body weight of females (related to lower body increment), serious changes in health condition status, higher incidence of the pathological findings in the dams, changes in reproduction parameters, and decreased number of live foetuses.
Executive summary:

Based on data from study, there is no need to update or change the classification and/or labelling of the substance.