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Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test results are not sufficient reported without details.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
not specified
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
corn oil
Details on exposure:
doses 0, 20, 40, 80 mg/kg bw/day dissolved in corn oil, exposure once daily
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
males: 49 days; females from 14 days before mating to day 3 of lactation
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0, 20, 40, 80 mg/kg bw/day dissolved in corn oil
Basis:
no data
No. of animals per sex per dose:
12 pre sex and group
Parental animals: Observations and examinations:
clinical signs of toxicity, body weight, mortality, pathology of male organs, number of pairs mated, number of pairs copulated, number of pregnant females, days until copulation, copulation index (no. of pairs with successful copulation / no. of pairs mated) X 100
fertility index (no. of pregnant rats / no. of pairs with successful copulation) X 100
duration of gestation,
---gestation index
(no. of females with live pups / no. of pregnant females) X 100,
---implantation index
(no. of implantations / no. of corpora lutea) X 100,
Oestrous cyclicity (parental animals):
estrus cycle length
Sperm parameters (parental animals):
no data
Litter observations:
live birth index (no. of live pups on day 0 / no. of pups born) X 100,
---delivery index (no of pups born / no. of implantations) X 100,
---sex ratio (no of males /no of females),
---viability index (no of live pups on day 4 / no. of live pups on day 0) X 100,
body weight of pups on day 0 and day 4
Postmortem examinations (parental animals):
Organ weight (absolute and relative):
- male, right and left testes, right and left epididymides
Statistics:
Statistical analysis of offspring was carried out using the litter as the experimental unit. Bartlett's test of homogeneity of variance was used to determine if the groups had equivalent at the 5% level of significance. If variance was equivalent, the groups were compared by one-way analysis of variance. If significant differences were found, Dunnett's test was performed. If the groups did not have equivalent variances, the Kruskal-Wallis test was used to assess the overall effects. Whenever significant differences were noted, Dunnett-type test was performed. Mann-Whitney U-test or Fisher's exact test was also used.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related
CLINICAL OBSERVATIONS AND FREQUENCY:
Mortality of two pregnantfemales was observed on day 21 or 22 day of pregnancy at a dose of 80 mg/kg bw/day.
Poor maternal behaviour and nursing were observed in 7 dams at a dose of 80 mg/kg bw/day. Gestation index was
slightly but not statistically significantly reduced in females pregnancy at a dose of 80 mg/kg bw/day: 90 % (1 of 10 surviving dams without
live pups) versus 100 % in controls. Reproductive parameters of animals at a dose of 40 mg/kg bw/day or less were unaffected by treatment with
Dicyclohexylamine. Reduction of ovary weights from 70 mg/kg bw/day dose level group upwards.

---Food consumption (graphic):
80 mg-groups of both sexes: low food consumption

PATHOLOGY
---male terminal mean body weight
low-mid-high dose versus control:
529g-547g-507g (p<0.05) versus 543g
---ORGANS EXAMINED AT NECROPSY (reported organs):
--Organ weight (absolute and relative):
- male, right and left testes: absolute weights showed no differences to concurrent controls
relative weights: low, mid, high dose versus control
- right testes: 0.32 g%, 0.31 g%, 0.34 g%(p<0.01) versus 0.30 g%
--left testes: no significant difference to the concurrent control
both testes together: 0.64 g%, 0.62 g%, 0.68g%(p<0.05) versus 0.61 g%
right and left epididymis: no significant differences to concurrent control

OTHER EXAMINATIONS:
---REPRODUCTIVE PERFORMANCE:
no differences to the concurrent controls:
-----estrus cycle length, number of pairs mated, number of -----pairs copulated, number of pregnant females, days -----until copulation, copulation index, fertility index
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no data
Remarks on result:
other: Generation: offsprings (migrated information)
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no data
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day
Sex:
male
Basis for effect level:
other: no data
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Sex:
female
Basis for effect level:
other: no data
Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
no differences to the concurrent controls:
---duration of gestation,
---implantation index
---delivery index
---sex ratio
findings which differ from controls:
low - mid - high dose versus control
---gestation index
100% - 100% - 90% versus 100%
1/10 surviving high dose dam without pups
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
40 mg/kg bw/day
Based on:
not specified
Sex:
male/female
Basis for effect level:
other: no data
Reproductive effects observed:
not specified
Conclusions:
NOAEL (offsprings): 40 mg/kg bw/day (male/female)
NOAEL (P): 40 mg/kg bw/day (male/female)
Executive summary:

Based on the Reproduction/Developmental toxicity screening test (OECD Guideline 421), NOAEL (offsprings): 40 mg/kg bw/day (male/female), NOAEL (P): 40 mg/kg bw/day (male/female)

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
40 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimish score 2 (test results are not sufficient reported without details)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Higher-tier fertility study (two-generation study) is not required, since there were no adverse effects observed in the 28-day and 90-day repeated dose toxicity studies on reproductive organs or tissues.

In an OECD reproduction/developmentaltoxicity screening test (OECD TG 421) in rats DCHA revealed effects on reproduction only in females at the highest oral dose tested (80 mg/kg bw/day) including slightly reduced gestation index, increase in stillborn pups and decrease in live born pups. Thus, the NOAEL (reproductive toxicity) was 80 mg/kg bw/day for males and 40 mg/kg bw/day for females. The NOEL (offspring) was 40 mg/kg bw/day based on significant reduction in pup weights on day 0 and slight reduction in pup weights on day offspring of the parents dosed with 80 mg/kg bw/day. These adverse effects on the development of the F1-generation occur only in the presence of severe maternal toxicity (17 % mortality; 1 of 10 surviving dams without live pups; poor maternal behavior and nursing).

Short description of key information:
The key information is based on the results from preliminary reproduction toxicity screening test by oral administration in rats performed according to OECD Test Guideline 421 Reproduction/developmental Toxicity Screening Test.
The NOAEL for reproduction was determined: 40 mg/kg bw/day for females; 80 mk/kg bw/day for males.

Justification for selection of Effect on fertility via oral route:
Only one study is available.

Effects on developmental toxicity

Description of key information
Based on  OECD Test Guideline No. 414 Prenatal Developmental Toxicity Study, the NOAEL (No Observed Adverse Effect Level) for prenatal development is higher than 160 mg/kg bw/day. 
Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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
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.

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
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.
Dose descriptor:
NOAEL
Effect level:
< 40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
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.
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)
Abnormalities:
not specified
Developmental effects observed:
not specified

For detailed information on results - see attached document.

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
160 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable without restriction.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity of Dicyclohexylamine was studied in prenatal developmental toxicity study in rats and resulted in NOAEL higher than 160 mg/kg bw/day for prenatal development. 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.


Justification for selection of Effect on developmental toxicity: via oral route:
Based on the Prenatal developmental toxicity using the Method B.31, Prenatal Developmental Toxicity Study, Council Regulation (EC) No. 440/2008,
and OECD Test Guideline No. 414 Prenatal Developmental Toxicity Study

Justification for classification or non-classification

Based on the test results and according to the EC criteria for classification and labelling requirements for dangerous substances and mixtures the test substance does not have to be classified for reproductive toxicity.