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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Remarks:
draft report
Adequacy of study:
key study
Study period:
17 November 2015 to 15 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
September 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
August 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclohexyldimethylamine
EC Number:
202-715-5
EC Name:
Cyclohexyldimethylamine
Cas Number:
98-94-2
Molecular formula:
C8H17N
IUPAC Name:
N,N-dimethylcyclohexanamine
Constituent 2
Reference substance name:
N,N-dimethylcyclohexylamine
Cas Number:
98-94-2
IUPAC Name:
N,N-dimethylcyclohexylamine
Constituent 3
Reference substance name:
N,N-dimethylcyclohexanamine
Cas Number:
98-94-2
IUPAC Name:
N,N-dimethylcyclohexanamine
Details on test material:
- Name of test material (as cited in study report): DMCHA
- Physical state: Clear, colourless liquid
- Purity test date: 99.4% w/w
- Lot/batch No.: 570071
- Expiration date of the lot/batch: 20 May 2011
- Molecular Weight: 127g/mol
- Molecular Formula: C8H17N
- Storage: At room temperature in the dark
- Stability: Stable
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Dimethylcyclohexylamine Batch No.: 1920179, obtained from Air Products and Chemicals, Inc. USA
- Expiration date of the lot/batch: 02/02/2018
- Purity test date: 99.3% w/w tested on 02/02/2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark; prepared dosing formulations were stored at 4°C in the dark
- Stability under test conditions: assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: stable in the vehicle (Arachis oil BP) for 14 days (determined during a previous study)


Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han:RccHan:WIST
Details on species / strain selection:
Species as recommended by the guideline, the strain is a commonly used laboratory strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: males 213 to 255 g; females 175 to 216 g
- Fasting period before study: not applicable
- Housing: same-sex groups of 3 or 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding with environmental enrichment (wooden chew blocks and cardboard fun tunnels)
- Diet (e.g. ad libitum): Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, ad libitum
- Water (e.g. ad libitum): mains drinking water ad libitum
- Acclimation period: at least 8 days

DETAILS OF FOOD AND WATER QUALITY: The diet, drinking water bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Certificates of analysis of the batches of diet used are available.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle (low intensity fluorescent lighting)

IN-LIFE DATES: From: 19 February 2016 To: 20 May 2016

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily, for up to ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe.
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: formulations were prepared weekly and stored at ~4°C in the dark. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): arachis oil BP was chosen based on previous studies
- Concentration in vehicle: 0, 2.5, 12.5, 37.5/25 mg/mL
- Amount of vehicle (if gavage): dose volumes were maintained at 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken on four occasions and analyzed for concentration of Dimethylcyclohexylamine at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations was GC using FID detection. The results indicate that the prepared formulations were within ± 8% of the nominal concentration.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Dose level was initially 150 mg/kg bw/d, but lowered to 100 mg/kg bw/d in females from Day 27 and in males from Day 28
No. of animals per sex per dose:
10 rats/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on previous studies conducted at the test facility with this test item
- Rationale for animal assignment: randomly allocated to treatment groups using a stratified body weight randomization procedure
Positive control:
No - not required

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, immediately post dosing and one hour after dosing. Due to clinical signs of toxicity being evident in animals of either sex treated at the high dose level, additional clinical observations were performed in these animals fifteen and thirty minutes after dosing from Days 27 (females) and 28 (males). Following the reduction of the dose
level and the regression of clinical signs, these additional observations were discontinued on Days 36 (females) and 37 (females). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights were recorded on Day 1 prior to dosing and at weekly intervals thereafter. Terminal body weights were also recorded.

FOOD CONSUMPTION: Yes
- Food consumption: recorded for each cage group at weekly intervals

FOOD EFFICIENCY: Yes
- Food conversion efficiency: calculated retrospectively using body weight and cage group food consumption

WATER CONSUMPTION: Yes
- Time schedule for examinations: water intake was observed daily for each cage group by visual inspection of the water bottles

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule and dose groups for examinations: the eyes of all control and treated animals were examined prior to treatment; the eyes of all control and high dose animals were examined before study termination (during Week 12). Examinations included observation of the anterior structures of the eye, and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl®), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Week 4 and at study termination (Day 90)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: 10/sex/dose
- Parameters examined: Haemoglobin (Hb); eryrocyte count (RBC); haematocrit (Hct); mean corpuscular haemoglobin (MCH); mean corpuscular volume (MCV); mean corpuscular haemoglobin concentration (MCHC); total leukocyte count (WBC); differential leukocyte counts - neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas); platelet count (PLT); prothrombin time (CT); activated partial thromboplastin time (APTT) . Reticulocyte count; methylene blue stained slides were prepared but not assessed.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Week 4 and at study termination (Day 90)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: 10/sex/dose
- Parameters examined: urea, glucose, total protein (Tot.Prot.), albumin, albumin/globulin (A/G) ratio (by calculation), sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca++), inorganic phosphorous (P), aspartate aminotransferase (ASAT), alamine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine (Creat), total cholesterol (Chol), total bilirubin (Bili), bile acids.

URINALYSIS: Yes
- Time schedule for collection of urine: during Week 4 and during the final week of treatment
- Metabolism cages used for collection of urine: Yes (overnight)
- Animals fasted: food was not available during urine collection
- Parameters examined: volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood, appearance.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Weeks 4 and 12, functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: all
- Battery of functions tested: behavioural assessment in purpose built arena; functional performance tests (motor activity and forelimb/hindlimb grip strength); sensory reactivity to auditory, visual and proprioceptive stimuli.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were subjected to a full external and internal examination, and any macropscopic abnormalities recorded.
- Organ weights were obtained for the following organs: adrenals, brain, epididymides (right and left separately), heart, kidneys, liver, ovaries, spleen, testes (right and left separately), thymus and uterus.

HISTOPATHOLOGY: Yes
- Samples of the following tissues were collected from all animals amd preserved in buffered 10% formalin, except where stated: adrenals, aorta (thoracic), bone and bone marrow (femur inlcuding stifle joint - retained only not processed), bone and bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, right epididymis (preserved in Modified Davidson's fluid), oesophagus, eyes (fixed in Davidson's fluid), gross lesions, heart, ileum (including Peyer's patches), jejunum, kidneys, liver, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin, spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, right testis (fixed in Davidson's fluid), thymus, thyroid/parathyroid, tongue (retained only not processed), lungs (with bronchi), lymph nodes (mandibular and mesenteric), mammary glands, muscle (skeletal - retained only not processed), trachea, urinary bladder, uterus (with cervix), vagina.
- All tissues from control and 150/100 mg/kg bw/day dose group animals and early decedents were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In addition, sections of the right testis from all control and 150/100 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Other examinations:
SPERM ANALYSIS
At necropsy, the left testis and epididymis were removed from all males, dissected from connective tissue and weighed separately. For the epididymis, the distal region was incised and a sample of the luminal fluid was collected and transferred to a buffer solution for analysis of sperm motility. The semen sample was assessed using an automated semen analyser to determine the numbers of motile, progressively motile and non-motile sperm. For the testis, the tunica albuginea was removed and the testicular tissue was stored frozen at approximately -20°C. The tissue was later thawed and homogenised in a suitable saline/detergent mixture. Samples of the homogenate were examined to determine the number of homogenisation resistant spermatids present. The cauda epididymis was separated from the body of the epididymis and weighed. The cauda epididymis was frozen at approximately -20°C. The tissue was later thawed and homogenised in an appropriate saline/detergent to determine the numbers of homogenisation resistant spermatids. Morphological assessment was performed on a sample of a minimum of 200 sperm, where possible, to determine the number with apparent structural anomalies. Assessment of homogenisation resistant spermatids and morphology was only performed for control and high dose males. As there were no treatment-related findings, these evaluations were not extended to males from other dose groups.
Statistics:
Data analysed using Provantis as follows: where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test.

Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights, Sperm Motility, Sperm Morphology and Homogenization Resistant Spermatids. Statistical significance was achieved at p<0.05.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Increased salivation was evident in surviving animals of either sex treated with 150/100 mg/kg bw/day from Day 17 (females) and Day 18 (males) onwards. Increased salivation was also evident to a lesser extent in animals of either sex treated with 50 mg/kg bw/day between Days 25 and 71 (males) and Days 22 and 43 (females). An isolated incident was also evident in one male treated with 10 mg/kg bw/day on Day 37. Two females treated with 150 mg/kg bw/day also showed incidences of hunched posture, lethargy, decreased respiratory rate and body tremors on Days 18 and 19. One of these females was also ataxic on Day 19.
The male treated with 150 mg/kg bw/day that was killed in extremis on Day 27 showed signs of increased salivation, vocalisation, body tremors, tonic convulsions, labored respiration and a decreased respiratory rate prior to termination. The female treated with 150 mg/kg bw/day that was found dead on Day 25 did not show any adverse clinical signs prior to death. The male treated with 150/100 mg/kg bw/day that was killed in extremis on Day 89 had an open wound. The control male that was killed in extremis on Day 62 showed signs of a stained snout, piloerection, dehydration, lethargy, pallor of the extremities, hunched posture and a decreased respiratory rate. Red staining was also observed in the cage of this animal on Day 61 only.
Mortality:
mortality observed, treatment-related
Description (incidence):
One male treated with 150 mg/kg bw/day was killed in extremis on Day 27. One female treated with 150 mg/kg bw/day was found dead on Day 25. No histopathological findings were evident in either animal which were considered to have contributed to the deaths. A further male from the 150/100 mg/kg bw/day dose group was killed in extremis on Day 89 due to a physical injury. This animal had a pituitary adenoma, correlating with enlargement at necropsy and which would have also contributed to its premature demise. In isolation, this was considered not to be a result of treatment. One female treated with 50 mg/kg bw/day died during the bleeding procedure on Day 90. In isolation, this was considered to be incidental. One control male was killed in extremis on Day 62 due to a decline in physical condition. No histopathological findings were evident in this animal which were considered to have contributed to the death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 150/100 and 50 mg/kg bw/day showed a statistically significant reduction (p<0.05-0.01) in body weight gain during the first week of treatment. Actual body weight losses were evident in the majority of females treated with 150/100 mg/kg bw/day. Recovery was evident in females treated with 150/100 mg/kg bw/day and in animals of either sex treated with 50 mg/kg bw/day thereafter, however, males treated with 150/100 mg/kg bw/day continued to show a statistically significant reduction (p<0.01) in body weight gain during Week 2 and a slight, but not statistically significant reduction, during Week 3. Overall body weight gain for these males was below controls, whilst corresponding value in females for this dose group was similar to controls. No such effects were detected in animals of either sex treated with 10 mg/kg bw/day.
Fluctuations in weekly group mean body weight gains were observed in males from all treatment groups during Weeks 6 and 7 and in females from all treatment groups during Week 13, achieving statistical significance (p<0.05-0.01). There was generally no dosedependence and these findings were considered to represent normal biological variation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 150/100 mg/kg bw/day showed a reduction in food consumption and food conversion efficiency during the first week of treatment. Males from this treatment group continued to show a reduction in food consumption during Week 2. Overall food consumption for animals of either sex treated with 150/100 mg/kg bw/day was lower than controls (17% for males and 22% for females). No effects were detected in animals of either sex treated with 50 or 10 mg/kg bw/day.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 150/100 mg/kg bw/day showed a reduction in food consumption and food conversion efficiency during the first week of treatment.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters examined. During Week 4 assessments, males from all treatment groups showed a statistically significant reduction (p<0.05-0.01) in total leucocyte and lymphocyte fraction counts. Animals of either sex treated with 150/100 mg/kg bw/day and females treated with 50 mg/kg bw/day showed a statistically significant reduction (p<0.01) in mean corpuscular hemoglobin concentration. Females treated with 150/100 mg/kg bw/day also showed a statistically significant reduction (p<0.05) in lymphocytes. During Week 13 assessments, males treated with 150/100 mg/kg bw/day showed a statistically significant reduction (p<0.01) in total leucocyte and lymphocyte fraction count. The majority of individual values were within historical background control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological importance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined. During Week 4 assessments, males treated with 150/100 mg/kg bw/day showed statistically significant reductions (p<0.05-0.01) in urea, total protein, potassium and phosphorus and a statistically significant increase (p<0.01) in albumin/globulin ratio. Males from all treatment groups showed a statistically significant reduction (p<0.05) in bilirubin. No such differences were evident in the corresponding females. During Week 13 assessments, animals of either sex from all treatment groups showed a statistically significant reduction (p<0.05-0.01) in urea and males from all treatment groups showed a statistically significant increase (p<0.05) in alkaline phosphatase. Males treated with 150/100 mg/kg bw/day also showed statistically significant reductions (p<0.05-0.01) in total protein and cholesterol. Females treated with 150/100 mg/kg bw/day also showed a statistically significant increase (p<0.05) in alkaline phosphatase and a statistically significant reduction (p<0.05) in aspartate aminotransferase with the effect onaspartate aminotransferase also extending to females treated with 50 mg/kg bw/day. The majority of individual values were within historical background control ranges and true dose related responses were not always evident. In the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological importance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Minor intergroup variations were evident in a few parameters measured however these did not follow a dose related response, were not consistent between sexes or did not have any associated renal microscopic changes evident and were therefore considered to represent normal biological variation.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no intergroup differences at any dose level considered to be related to treatment with the test item. During Week 4 assessments, a statistically significant reduction in hind limb grip strength was evident in males treated with 150/100 mg/kg bw/day when compared with controls (p<0.05). A statistically significant reduction (p<0.05) in overall activity was also evident in males from all treatment groups. Females from all treatment groups showed a statistically significant increase in overall activity (p<0.01) and the final 20% activity (p<0.05). There was no dose relationship throughout the treatment groups and the finding in hind limb grip strength was only evident in one out of the three tests. Therefore in the absence of any similar effects during the Week 12 assessments the intergroup differences were considered to be unrelated to treatment with the test item. During the Week 12 assessments, males treated with 150/100 mg/kg bw/day showed a
statistically significant reduction (p<0.01) in the final 20% activity whilst females from this treatment group showed a statistically significant increase (p<0.05) in fore limb grip strength. In the absence of any associated clinical signs of neurotoxicity the intergroup differences were considered to be unrelated to treatment with the test item.
Sensory reactivity scores across all test item-treated dose groups were similar to controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the organ weights measured. Males treated with 150/100 mg/kg bw/day showed a statistically significant reduction (p<0.05) in absolute kidney weight and a statistically significant increase (p<0.05) in relative (to terminal body weight) kidney weight. Males from all treatment groups also showed a statistically significant reduction (p<0.05-0.01) in thymus weight both absolute and relative to terminal body weight. Most individual values were within the historical control data ranges and a true dose related response for relative weights was not evident. In the absence of any histopathology correlates, these findings were considered not to be of any toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no macroscopic abnormalities evident in surviving animals that were considered to be treatment-related. The male treated with 150/100 mg/kg bw/day that was killed in extremis on Day 27 had reddened lungs and the male from this treatment group that was killed in extremis on Day 89 had a mass on the pituitary. The female that was found dead on Day 25 did not show any macroscopic abnormalities. The female treated with 50 mg/kg bw/day that died during the bleeding process on Day 90 had dark kidneys and red lungs. The control male that was killed in extremis on Day 62, had the majority of tissues recorded as pale, a mottled liver and dark colored contents in the intestines.
Three control females, two males and two females treated with 10 mg/kg bw/day, four males and three females treated with 50 mg/kg bw/day and one male and two females treated with 150/100 mg/kg bw/day had reddened lungs at necropsy. In the absence of any dose relationship or histopathological correlates the intergroup differences were considered not to be of toxicological importance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse effects on sperm concentration, motility, progressive motility, morphology or homogenization resistant spermatid counts were observed in treated males when compared to controls. A statistically significant increase (p<0.001) in homogenization resistant spermatid counts was evident in males treated with 150/100 mg/kg bw/day. No histopathological correlates were evident in the testes or epididymides and an increase in this parameter is considered not to represent an adverse effect of treatment.
Details on results:
See above

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Remarks on result:
not measured/tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Group mean body weights as study start and termination

Time point

Mean body weights (g) ± SD

0 mg/kg bw/d (control)

10 mg/kg bw/d

50 mg/kg bw/d

150/100 mg/kg bw/d

Males

 Day 1

239.3 ± 8.6

(n = 10)

231.2 ± 9.9

 (n = 10)

229.8 ± 7.2

(n = 10)

228.1 ± 10.8

(n = 10)

Day 91

432.3 ± 40.5

(n = 9)

416.5 ± 20.7

(n = 10)

414.4 ± 19.0

(n = 10)

382.4 ± 23.3

(n = 8)

Females

 Day 1

190.8 ± 9.9

(n = 10)

190.1 ± 9.0

 (n = 10)

189.5 ± 6.9

(n = 10)

186.7 ± 5.6

(n = 10)

Day 91

241.6 ± 16.7

(n = 10)

257.2 ± 22.7

(n = 10)

243.3 ± 15.5

(n = 9)

240.4 ± 17.1

(n = 9)

Applicant's summary and conclusion

Conclusions:
The NOAEL was considered to be 100 mg/kg bw/d in males and females.
Executive summary:

The potential for dimethylcyclohexylamine to cause systemic toxicity following repeated oral administration was investigated in the rat (according to OECD 408). The test item was administered by gavage to groups of 10 male and 10 female Wistar Han:RCCHan:WIST rats at doses of 0 (vehicle control), 10, 50 and 150 mg/kg bw/day (reduced to 100 mg/kg bw/day on Day 27 for females and Day 28 for males) for up to 90 consecutive days. Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all animals during Week 4 and at the end of the study. Ophthalmoscopic examination was also performed on control and treated groups prior to the start of treatment and on control group and high dose animals during Week 12. All animals were subjected to gross necropsy examination, sperm analysis and histopathological evaluation of selected tissues from high dose and control animals was performed.

One male treated with 150 mg/kg bw/day was killed in extremis on Day 27. One female treated with 150 mg/kg bw/day was found dead on Day 25. A further male from this treatment group was killed in extremis on Day 89 due to a physical injury. One female treated with 50 mg/kg bw/day died during the blood sampling procedure on Day 90 (in isolation this was considered to be incidental). One control male was killed in extremis on Day 62 due to a decline in physical condition; at necropsy the majority of tissues were recorded as pale. There were no further unscheduled deaths. The male treated with 150 mg/kg bw/day that was killed in extremis on Day 27 showed signs of increased salivation, vocalisation, body tremors, tonic convulsions, laboured respiration and a decreased respiratory rate. The female treated with 150 mg/kg bw/day that was found dead on Day 25 did not show any adverse clinical signs prior to death and no abnormalities were noted at necropsy. The male treated with 150/100 mg/kg bw/day that was killed in extremis on Day 89 had an open wound, and a mass on the pituitary was found at necropsy. The control male that was killed in extremis showed signs of a stained snout, pilo-erection, dehydration, lethargy, pallor of the extremities, hunched posture and a decreased respiratory rate. Two surviving females treated with 150 mg/kg bw/day showed incidences of ataxia, hunched posture, lethargy, decreased respiratory rate and tremors on Days 18 and 19. Increased salivation was evident in surviving animals of either sex treated with 150/100 mg/kg bw/day and to a lesser extent in animals of either sex treated with 50 mg/kg bw/day throughout the treatment period. An isolated incident was also evident in one male treated with 10 mg/kg bw/day on Day 37.

Animals of either sex treated with 150/100 and 50 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Actual body weight losses were evident in the majority of females treated with 150/100 mg/kg bw/day. Recovery was evident in females treated with 150/100 mg/kg bw/day and in animals of either sex treated with 50 mg/kg bw/day thereafter, however, males treated with 150/100 mg/kg bw/day continued to show a reduction in body weight gain during Weeks 2 and 3 and overall gain for these males was below controls. No such effects were detected in animals of either sex treated with 10 mg/kg bw/day. Animals of either sex treated with 150/100 mg/kg bw/day showed a reduction in food consumption and food conversion efficiency during the first week of treatment. Males from this treatment group continued to show a reduction in food consumption during Week 2. Overall food consumption for animals of either sex treated with 150/100 mg/kg bw/day was lower than controls. No such effects were detected in animals of either sex treated with 50 or 10 mg/kg bw/day.

There were no treatment-related effects on measured behavioural parameters, functional performance or sensory reactivity. Opthalmoscopic examination of animals of both sexes from the control and 150/100 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatmentrelated differences. There were no treatment-related effects on examined haematological, clinical chemistry and urinalysis parameters. There were no treatment-related abnormalities noted at necropsy, no effects on organ weights and no microscopic abnormalities were detected. No adverse effects on sperm concentration, motility, progressive motility, morphology or homogeneity resistant spermatid counts were observed in treated males when compared to controls.

Treatment with the test item resulted in the death of two animals treated with 150 mg/kg bw/day, clinical signs of toxicity and reduced body weight gains and food consumption at the high dosage group. No adverse effects were evident at 50 or 10 mg/kg bw/day. The clinical signs of toxicity and reduced body weight gain/food consumption was considered to be the result of the administration of the higher dose level (150 mg/kg bw/day) and once the dose level had been reduced recovery was evident. For these reasons, 100 mg/kg bw/day was regarded as the NOAEL for animals of either sex.