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Description of key information

Repeated Dose 90-day Oral Toxicity Study in Rodents performed according to EU Method B.26 was taken as the key study, resulting in NOAEL of 10 mg/kg body weight/day for males and females. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16.4.-9.10.2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
See Overall remarks
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar CRL
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Únětice, Czech Republic
- Age at study initiation: 7-8 weeks
- Weight at study initiation: approx. 190-215 g (males), 175-200 g (females)
- Housing: SPF animal house, 2 rats of the same sex in one plastic cage containing sterilized Lignocel
- Diet (e.g. ad libitum): complete pelleted diet for rats in SPF breeding - ST 1
- Water (e.g. ad libitum): Free access to drinking water
- Acclimation period: 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
Start of administration: 22. 04. 2014
End of administration: 23 07. 2014
Clinical examinations: main groups 22. 4. – 24. 7. 2014, satell.groups 22. 4. – 22. 7. 2014
Urinalysis: main groups 21. – 24. 7. 2014, satell.groups 18. – 19. 8. 2014
Blood taking and necropsies: main groups 22. – 25. 7. 2014, satell.groups 19. – 20. 8. 2014
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was weighted into a 150mL glass beaker calibrated to 100 mL and the beaker was replenished by the vehicle and dissolved in ultrasonic bath for 5 min. The solution was stirred by magnetic stirrer (800 rpm) for 15 minutes. Two concentrations of application form were prepared (2200 mg/100 mL and 100 mg/100 mL).

VEHICLE
- Justification for use and choice of vehicle (if other than water): test substance insoluble in water
- Amount of vehicle (if gavage): 1 mL of suspension per 100 g of body weight
- Lot/batch no. (if required): 5464101, 5223502

STABILITY AND HOMOGENITY OF THE PREPARATION: It follows from results of analyses for concentration level 2200 mg/100 mL (homogeneity and stability) that the solution of the test substance, Dicyclohexylamine, in vehicle at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 120 minutes from a finalization of the application form preparation.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
10 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
90 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 females and 10 males
6 females and 6 males (satallite groups)
Control animals:
yes, concurrent vehicle
other: satellite group - control - vehicle... (see attached file)
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, the following doses - 0, 10, 30 and 90 mg/kg bw/day were chosen for Repeated Dose 90-day Oral Toxicity Study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice a week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: at the 1st week of study and at the last week of administration and recovery period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 91st (main group); 119th (satellite group) day of study
- Anaesthetic used for blood collection: Yes - light ether narcosis
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table No.2 were examined.

BIOCHEMICAL EXAMINATION: Yes
- Time schedule for collection of blood: 91st (main group); 119th (satellite group) day of study
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table No.3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: 90th (main group); 118th (satellite group) day of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table No.4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the last week of administration and recovery period
- Dose groups that were examined: all groups
- Battery of functions tested: sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex, motor activity, individual observations of grip strength on pectoral legs and pelvis legs.
Statistics:
For statistical evaluation the software Statgraphic ® Centurion (version XV, USA) was used.
Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group.
The results statistically significant on probability level 0.05 are indicated in the summary tables.

PARAMETRIC TESTS
As the first step the test for normality (Shapiro-Wilk test) was used. When data were not normally distributed than transformation of data was performed (Box-Cox transformation). Due to still not normal distribution the non-parametric methods were used for further analysis. At first it was the Kruskal-Wallis test (for satellite groups only this one test) for the comparison of the measured effect in all treatment groups with the vehicle control group, as global test, and then, in case of statistical significant findings, the two-groups Mann-Whitney test (probability level 0.05) was used.
If data were normally distributed the variance check (Levene´s test) was used to verify if standard deviations within each group are equal. One-way ANOVA (probability level 0.05) was used to detect whether there are any significant differences amongst the means. If significant differences were found the post hoc statistical testing was performed (Fisher's least significant difference - LSD test). If was worried about the presence of outliers, the appropriate non-parametric test (Kruskal-Wallis Test, Mann-Whitney test) was chosen.

NON PARAMETRIC TESTS
At first the Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group, as global test, and then in case of statistically significant findings the two-groups Mann-Whitney test (probability level 0.05) was used.

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Slight disbalance of food consumption and/or food conversion was recorded in both sexes.
Food efficiency:
no effects observed
Description (incidence and severity):
Slight disbalance of food consumption and/or food conversion was recorded in both sexes.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Slight disbalance of water consumption was recorded in both sexes.
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Description (incidence and severity):
Changes of urine parameters were reversible and without toxicological importance.
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
The oral administration at the lowest and middle dose levels did not cause mortality. At the highest dose level one male died on the 50th day of study and one female was humanely killed by reason of moribundus status on the 43th day of study.
Clinical observation revealed influence of the test substance on clinical status of treated animals. Significant findings mainly convulsions and salivation was observed. The convulsions were often accompanied by marked salivation and occasionally it was inwoked by external stimulation like presence of person performing clinical observation, changing of bedding etc. These changes of clinical status showed influence of the test substance on nervous system but histopathological changes of nervous system organs were not found out.
Hunched posture, vocalisation, piloerection, apathy, irritability, dirty fur, creaky respiration was also recorded in treated animals of both sexes. These changes of clinical status were with toxicological importance and related with application of the test substance. After the end of application of the test substance all clinical symptoms disappeared.

BODY WEIGHT AND WEIGHT GAIN
Effect of the test substance administered at the highest dose level on growth of animals was observed. In satellite females significant decrease of body weight was recorded from the 8th week of application to the end of recovery period. In males slightly decreased body weight was observed.

OPHTHALMOSCOPIC EXAMINATION
Functional and ophthalmological examination mydriasis and slowed-down pupillary reflex in animals of both sexes reveated. These changes of eyes related with the test substance administration but they were reversible and during recovery period these clinical symptoms disappeared.

HAEMATOLOGY
Haematological examination showed reversible effect on blood component mainly in females – total erythrocyte count and haemoglobin concentration was significantly increased at the highest dose level. Significantly increased value of haematocrite at the middle and highest dose level was also recorded and insignificantly it was recorded at the lowest dose level. It was dependent on dose level. Diagnosed polycytemia was probably relative: an apparent rise of the erythrocyte level in the blood and elevated haematocrit; however, the underlying cause could be reduced blood plasma. In addition the density of urine was also increased (see below). In white blood component total leucocyte count in females at the highest dose level was recorded. Leucocytosis could relate with occurrence of convulsion which was recorded during clinical observation. Following significant changes in haemocoagulation parameters were recorded: shortened of APTT and prothrombin time at the highest dose level in females and decreased value of platelet count at the lowest dose level and shortened of APTT at the middle and highest dose levels in males. These changes were reversible. After recovery period only value of prothrombin time in females was changed.

CLINICAL CHEMISTRY
Biochemical examination of blood revealed statistically significant changes in all trated groups. In males the following significant changes were detected: decreased value of sodium ions in all treated groups (with dependendence on dose level), increased value of total protein at the lowest and middle dose level, increased activity of AST and decreased value of bilirubin total at the highest dose level, decreased value of glucose at the middle dose level and decreased value of urea and increased value of calcium ions at the lowest dose level 10 mg/kg/day. Biochemical changes of bilirubin and activity of AST could be related with microscopical changes of liver (see above). In males also delayed increase of sodium ions value and value of inorganic phosphorus and decrease of value of protein total was detected. In females the following significant canges were registered: decreased value of creatinine in all treated groups, increased activity of AST (it was irreversible), increased value of urea and concentration of phosphorus ions, decreased value of protein total, albumin and concentration of sodium ions. Delayed increase of value of glucose was also detected in females.

URINALYSIS
During urinalysis significant increase of urine pH was detected in females at the middle dose level and in males at the highest dose level. At the highest dose level occurrence of protein in one male and change of colour of urine in one male was detected. Occurrence of leucocytes in urine was recorded mainly in males. Urine specific gravity in both sexes was increased with dose dependence. Changes of urine parameters were reversible and without toxicological importance.

NEUROBEHAVIOUR
Slowed-down pupillary reflex in animals of both sexes reveated.

ORGAN WEIGHTS
During inspection of biometry of organs significant changes were recorded. Decreased absolute and relative weight of thymus in males at the highest dose level was recorded. Relative and absolute weight of adrenal glands at the highest dose level in both sexes was increased. No microscopical changes of adrenal glands was detected. In males of all treated groups increase of relative weight of liver was detected (in the middle and highest dose levels with statistical significance). Changed weight of liver in males could relate vacuolation in liver. Occurrence of vacuolation of liver in males was detected in all groups incl. control but in treated animals foci of hepatocytes with vacuolation were more extensive. These changes was reversible and probably it was adaptation reaction of organism to the test substance treatment. Presence of fatty droplets in hepatocytes of treated males was confirmed durig microscopical examination of specially stained samples of liver. In females occurrence of vacuolations in liver was insignificant.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopical changes (vacuolation in liver, oedema in submucosa of stomach in males) - at the dose level 10 mg/kg/day.
Microscopical changes (vacuolation in liver, oedema in submucosa of stomach in males and hydronephrosis in kidneys in females) - at the dose level 30 mg/kg/day.
Microscopical changes (vacuolation in liver, oedema in submucosa in stomach and forestomach in males) - at the dose level 90 mg/kg/day.

Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

For detailed information on results - see attached document

Conclusions:
The oral administration of Dicyclohexylamine to rats by gavage for a period of 90 consecutive days at the highest dose level caused mortality The one male died on the 50th day of study and one female was humanely killed by the reason of moribundus status on the 43th day of study.
The test substance caused significant changes of clinical status of animals (mainly convulsions accompanied with marked salivation). These clinical findings were detected in both sexes at the highest dose level. At the middle dose level these symptoms were recorded only sporadically and at the lowest dose level only salivation in males was observed. These changes of clinical status were with toxicological importance and related with application of the test substance. After the end of application of the test substance all clinical symptoms disappeared.
During haematological examination changes of blood component were recorded: in males decreased value of APTT at the highest and middle dose level, and decreased value of platelet count at the lowest dose level and in females increased value of haematocrite in all treated groups, increased value of total erythrocyte count, haemoglobin at the highest dose level. These changes related with the test substance treatment but they were irreversible. In females at the highest dose level decreased value of platelet count was also detected.
During biochemical examination the following changes were detected: in males decreased concentration of sodium ions in all dose levels; on the contrary delayed increased aktivity of sodium ions, increased value of protein total at the lowest and middle dose level and on the contrary delayed decreased value of protein total, increased value of bilirubin and decreased aktivity of AST at the highest dose level and in females increased value of protein total, albumin and sodium ions.
During biometry of organs changes of weight of adrenal glands and thymus at the lowest and highest dose level, increased weight of liver at the middle and highest dose level in males and irreversible increased weight of adrenal glands in females were recorded. Toxicologically significant microscopical changes of these organs were not found out.
Urine specific gravity in both sexes (at the middle and highest dose level) was increased with the dose dependence. Changes of urine parameters were reversible and without toxicological importance.
The test substance had influence on microscopical structure of liver and stomach (vacuolations and focal fatty changes mainly in males and oedema in stomach). Observed local microscopical changes were probably caused by application of the test substance but they were without toxicological importance and had only adaptation character.
Executive summary:

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimish score 1 (GLP compliance, no deviations)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

90-day repeated oral dose toxicity study in rats resulted in NOAEL of 10 mg/kg body weight/day based on clinical observation (mainly salivation and convulsion), haematological examination (decreased value of platelet count nad APTT in males and increased value of hematocrite, total erythrocyte count, heamoglobine and leucocyte count and decreased value of platelet count in females), biochemical examination (increased value of protein total, decreased value of sodium ions in males and decreased value of sodium ions and protein total, irreversible increased value of AST in females) and biometry of organs results in males (weight of thymus, adrenal glands and liver). These effects were considered not to support the classification of substance for specific target organ toxicity but are taken into account in risk assessment. In case of Repeated dose toxicity (dermal and inhalation route) dose descriptors are based on extrapolation from oral study.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study with the longest duration (90-days) and lowest NOAEL was chosen.

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 repeated dose toxicity. Effects observed in repeated dose 90-day oral toxicity study were considered and they do not support classification for specific target organ toxicity following repeated exposure to DCHA.