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Administrative data

Description of key information

The repeated dose toxicity of the substance was investigated in sub-acute studies in rats using inhalation, gavage and dietary exposure.  The results of these studies demonstrate an absence of systemic toxicity and show that the primary effects of exposure to the test material are local and attributable to its corrosive nature.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
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
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)


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
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
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
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.
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
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
Key result
Critical effects observed:
no

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)

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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
IN-LIFE DATES: From: September 30th, 2009 To: December 7th, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out according to OECD guideline 422 and follows the GLP compliance.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 12 weeks
- Weight at study initiation: Males: 297 - 303 g; Females: 200 - 204 g
- Fasting period before study: Not applicable
- Housing: Housed in a controlled environment in Macrolon cages (MIV type, height 18cm). In the pre-mating period, animals were housed 5 animals/sex/cage.
- Diet (e.g. ad libitum): Free access to prepared diets, Standard powder rodent diet (SM R/M-Z fromSSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): Free access to tap-water
- Acclimation period: At least 5 days prior to start of treatment.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C (actual range: 20.1 – 21.8°C)
- Humidity (%): 40 - 70% (actual range: 32 - 100%)
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day


IN-LIFE DATES: From: September 30th, 2009 To: December 7th, 2009
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test substance.

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared once weekly
- Mixing appropriate amounts with (Type of food): powder feed (premix) and subsequently mixed with the bulk of the diet.
- Storage temperature of food: Kept at room temperature in the diet store room

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy, homogeneity and stability were determined for diets prepared for use during treatment.
For determination of accuracy, samples were taken at random position or at 90%, 50% and 10% height. The latter set of samples was also used for the determination of the homogeneity of the diets. For determination of stability, additional samples were taken at 50% height and stored at room temperature for 2 weeks or 8 days. Analyses were performed on samples taken in week 4 and week 7. Analysis was carried out using LC-MS/MS.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for
41-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 5 days of lactation.

Frequency of treatment:
Ad libitum for at least 28 days. Animals received test diet from Day 1 until the day prior to necropsy.
Dose / conc.:
0 ppm
Dose / conc.:
150 ppm
Remarks:
nominal in diet
Dose / conc.:
500 ppm
Remarks:
nominal in diet
Dose / conc.:
1 500 ppm
Remarks:
nominal in diet
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, plain diet
yes, historical
Details on study design:
- Dose selection rationale: The dietary inclusion levels were based on the results of the dose range finding study. Rats were exposed to 500, 1500 and 5000 ppm. At 5000 ppm, severe reduction in food consumption, body weight loss and hunched posture were reported. At 500 and 1500 ppm, reduction in food consumption with slight receovery was noted. On this basis, 1500 ppm was selected as the highest dose to be tested.
- Rationale for animal assignment (if not random): Not applicable
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not applicable
Positive control:
Not applicable
Observations and examinations performed and frequency:
MORTALITY/VIABILITY: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily (Observations were also made outside the cage prior to start treatment and at weekly intervals thereafter)

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly for males and females. During the mating period, food consumption in males was recorded on Days 1, 8 and 14. After mating, food consumption in males was recorded on days 8 and 14. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on days 1 and 4 of lactation. In non-mated females, food consumption was recorded at least once weekly manually after completion of the mating period until necropsy.
- Compound intake: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes, subjective appraisal was performed during the study.

OPHTHALMOSCOPIC EXAMINATION: Not performed as it is not required in the OECD guideline 422

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Taken prior to necropsy
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, animals were fasted overnight (for a maximum of approximately 20 hours).
- How many animals: 5 animals/sex/dose group
- Parameters checked: Haematology: White blood cells, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, Clotting Potential: Prothrombin time, activated Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Taken prior to necropsy
- Animals fasted: Yes, animals were fasted overnight (for a maximum of approximately 20 hours).
- How many animals: 5 animals/sex/dose group
- Parameters checked: Clinical Biochemistry: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, bile acids

URINALYSIS: Not performed since it is not mandatory according to OECD guideline 422.

NEUROBEHAVIOURAL EXAMINATION: Yes,
- Time schedule for examinations: 5 males/ dose group were tested during Week 4 of treatment and 5 females/dose group were tested during lactation
- Dose groups that were examined:All dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity


OTHER: General reproduction data includiing male number paired with, mating date, confirmation of pregnancy and delivery day were also recorded.
Sacrifice and pathology:
Termination was scheduled for females which delivered on days 5-7 of lactation, females which failed to deliver post-coitum day 26 and 21 days after the last day of the mating period. Males were necropsied after completion of the mating period (at least 28 days after dose administration)

GROSS PATHOLOGY: Yes, 5 animals/ sex/group
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs.

The following organs were examined with the exception of the tissues/organ in parentheses for which no signs of toxicity were noted at macroscopic examination.

Identification marks: not processed
Adrenal glands
Aorta
Brain (cerebellum, mid-brain, cortex)
Caecum P
Cervix
Clitoral gland
Colon
Duodenum
Epididymides
Eyes with optic nerve (if detectable) and
Harderian gland
(Female mammary gland area) Spinal cord -cervical, midthoracic, lumbar
Femur including joint
Heart
Ileum
Jejunum
Kidneys
(Larynx)
(Lacrimal gland, exorbital)
Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
(Nasopharynx)
Oesophagus
Ovaries
Pancreas
Peyer's patches (jejunum, ileum) if detectable
Pituitary gland
Preputial gland
Prostate gland
Rectum
(Salivary glands - mandibular, sublingual)
Sciatic nerve
Seminal vesicles including coagulating glands
Skeletal muscle
(Skin)
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes
Thymus
Thyroid including parathyroid (if detectable)
(Tongue)
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions

All remaining animals and females which failed to deliver 2:
Cervix
Clitoral gland
Coagulation gland
Epididymides
Ovaries
Preputial gland
Identification marks: not processed
Prostate gland
Seminal vesicles
Testes
Uterus
Vagina
All gross lesions

HISTOPATHOLOGY: Yes
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands) in 5 males from the control and high dose group. Additional slides of the testes of 5 males of group 1 and 4 were also examined. All gross lesions of all dose groups and the reproductive organs of all animals that failed to mate, conceive, sire or deliver healthy pups were also studied.



Other examinations:
Organ weights were recorded in 5 animals/sex/group:
Adrenal glands, Brain , Epididymides , Heart , Kidneys , Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix), Prostate, Seminal vesicles including coagulating gland, Thyroid including parathyroid
All remaining males:
Epididymides + Testes
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.
The number of corpora lutea was transformed by using 1/x to obtain a normal distribution. This was followed by ANOVA. The Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
The number of implantation sites was subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal, 1952) to determine intergroup difference. If the results of the ANOVA were significant (p<0.05), the Wilcoxon test (Wilcoxon, 1945) was applied to the data to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Test statistics were calculated on the basis of exact values for means and pooled variances.
Individual values, means and standard deviations may have been rounded off before printing.
Therefore, two groups may display the same printed means for a given parameter, yet display
different test statistics values.
No statistical analysis was performed on histopathology findings.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see results below
Mortality:
mortality observed, treatment-related
Description (incidence):
see results below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see results below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see results below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see results below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see results below
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
see results below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see results below
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see results below
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see results below
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see results below
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.
No clinical signs of toxicity were noted during the observation period.
Slight alopecia was noted for one female rat of Group 1. This finding occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study.

BODY WEIGHT AND WEIGHT GAIN
Body weight and body weight gain was reduced for males and females on Day 8 prior to mating in the 1500 ppm dose group. This was considered to be related to the decreased food consumption in these animals on Days 1-8. Body weights and body weight gain remained lower in these animals
during the mating period; however when body weight gain was calculated from Day 8 onwards no difference was noted compared to the control group.
In females treated at 500 and 1500 ppm decreased body weights and body weight gain (not always statistically significant) were noted during the post-coitum phase. However, the decrease was not always statisticalyl significant. In these females, body weights were also reduced during the lactation phase.

Terminal body weight was significantly lower in males at 1500 ppm and females at 500 and 1500 ppm when compared to control animals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption (absolute and relative) was lower for males and females treated at 1500 ppm over Days 1-8 premating, with recovery of food consumption after Day 8. Slightly reduced food consumption was also noted for females treated at 1500 ppm on several occasions post-coitum, and during lactation. Minor statistically significant differences arising between controls and females receiving 150 or 500 ppm during post-coitum were considered not to represent a change of biological significance.

HAEMATOLOGY
No changes in haematological parameters were reported. Changes that occurred were considered to be of no toxicological relevance.

CLINICAL CHEMISTRY
No changes in clinical parameters were deeemed to be treatment related. One male at 1500 ppm had decreased aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), albumin and chloride, and increased cholesterol, urea, and creatinine. Total bilirubin was statistically significantly increased for low and high dose females when compared to controls. These changes were considered to have arisen as a result of slightly low control values and in the absence of a dose-response relationship were considered to be of no toxicological significance.

ORGAN WEIGHTS
The following statistically significant changes in organ weights distinguished treated from control
animals:
- Decreased absolute prostate weights at 1500 ppm
- Decreased absolute and relative seminal vesicles weights at 1500 ppm
- Decreased absolute thymus weights for females at 500 and 1500 ppm
- Decreased absolute adrenals weights for females at 1500 ppm
- Decreased absolute spleen weights for females at 1500 ppm
In the absence of any corroborative microscopic findings, these changes were considered to be of no toxicological significance.

High weights for liver and kidneys and low weight for the testes were recorded in one male in the 1500 ppm dose group. These findings were in line with the enlarged liver and kidneys and reduced size of the testes observed macroscopically.

Increased relative brain weights in males in the 1500 ppm dose group and in females in the 500 and 1500 ppm dose group were not deemed to be of toxicological relevance.

The statistical significant changes noted for w eights of ovaries and uteri were considered to be caused by the relatively high control value. All other statistical significant changes (thyroids of males at 150 and 500 ppm and spleen of females at 500 ppm) were in the normal range of biological variation noted for rats of this strain and age. In the absence of a dose-response relationship, they were considered to be of no toxicological significance.

GROSS PATHOLOGY
No macroscopic changes at necropsy were deemed to be treatment related. Changes such as enlarged liver and kidneys, pelvic dilation and pale discolouration of the kidneys and reduced size of the testes were reported in one male in the 1500 ppm dose group. One female in the 150 ppm dose group showed an enlarged spleen and a soft red-brown nodule in the subcutis of the genital region. Incidental findings were also noted however these findings were within the hitorical contriol range among rats of this age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment related microscopic findings were reported. One male at 1500 ppm showed slight centrilobular
hepatocellular liver hypertrophy; marked, bilateral, progressive nephropathy and slight vacuolation of the zona fasciculata of the adrenal glands. These pathologic changes were considered to be part of an underlying systemic defect. These findings were not deemed to be treatment related since none of the other rats in this group showed any overt kidney, liver or
adrenal gland toxicity

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
One female in the 150 ppm dose group displayed a mammary gland adenocarcinoma. However, this is a common finding in this strain of rat of this age.

OTHER FINDINGS
No abnormalities were seen in the reproductive organs of suspected non-fertile animals which could account for infertility.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
No toxicologically significant effects on reproductive parameters were noted.
Dose descriptor:
NOAEL
Remarks:
correced for mean test article intake
Effect level:
>= 91 - <= 104 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: No effects were reported to be treatment related.
Key result
Dose descriptor:
NOAEL
Remarks:
corrected for mean test article intake
Effect level:
>= 85 - <= 147 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: No effects were reported to be treatment related
Key result
Critical effects observed:
no

Table 1: Summary of body weights in males

Day

0 ppm

(g)

150 ppm

(g)

500 ppm

(g)

1500 ppm

(g)

Pre mating

 

 

 

 

Day 1 (Week 1)

302 ± 14.7

303 ± 11

303 ± 15.4

297 ± 9.7

Day 8 (Week 2)

324 ± 18.1

325 ± 12.8

322 ± 17.6

302** ± 12.3

Mating period

 

 

 

 

Day 1 (Week 1)

341 ± 20.6

343 ± 16.2

338 ± 17.2

316**± 18.2

Day 8 (Week 2)

347 ± 20.8

352 ± 21.0

346 ± 19.5

319* ± 20.1

Day 14 (Week 2)

358 ± 21.0

364 ± 23.0

353 ± 18.6

329* ± 25.3

 

Table 2: Summary of body weights in females

Day

0 ppm

(g)

150 ppm

(g)

500 ppm

(g)

1500 ppm

(g)

Pre mating

 

 

 

 

Day 1 (Week 1)

201 ± 5.9

204 ± 7.4

200 ± 4.8

204 ± 5.0

Day 8 (Week 2)

209 ± 8.2

210 ± 7.6

203 ± 4.6

197** ± 4.1

Mating period

 

 

 

 

Day 1 (Week 1)

214 ± 6.5

215± 8.7

208* ± 4.8

202** ± 3.1

Day 8 (Week 2)

236a

-

225a

216 ± 2.8b

Day 14 (Week 2)

235a

-

-

218a

Day 22 (week 4)

234a

-

-

229a

Day 29 (week 5)

235a

-

-

216a

aonly one female was weighed

btwo females were weighed

 

Table 3: Summary of bodyweight gain (%) in males

Day

0 ppm

(g)

150 ppm

(g)

500 ppm

(g)

1500 ppm

(g)

Pre mating

 

 

 

 

Day 1 (Week 1)

0

0

0

0

Day 8 (Week 2)

7 ± 1.2

7 ± 0.8

6 ± 1.1

2** ± 2.4

Mating period

 

 

 

 

Day 1 (Week 1)

13 ± 2.1

 13 ± 1.6

12± 1.4

6** ± 4.5

Day 8 (Week 2)

15 ± 2.1

16 ± 3.4

14 ± 2.0

7** ± 5.0

Day 14 (Week 2)

19 ± 2.3

20 ± 3.7

17 ± 2.7

11** ± 6.7

 


 

Table 4: Summary of bodyweight gain (%) in females

Day

0 ppm

(g)

150 ppm

(g)

500 ppm

(g)

1500 ppm

(g)

Pre mating

 

 

 

 

Day 1 (Week 1)

0

0

0

0

Day 8 (Week 2)

4 ± 2.8

3 ± 2.4

2 ± 1.7

-3** ± 1.8

Mating period

 

 

 

 

Day 1 (Week 1)

7 ± 2.5

5 ± 2.9

4* ± 2.5

-1** ± 2.2

Day 8 (Week 2)

17a

-

8a

5 ± 1.4b

Day 14 (Week 2)

16a

-

-

6a

Day 22 (week 4)

16a

-

-

11a

Day 29 (week 5)

16a

-

-

5a

aonly one female was weighed

btwo females were weighed

Conclusions:
Changes in bodyweight and food consumption at 500 ppm and 1500 ppm were not deemed to be due to DMCHA.
Based on the absence of treatment related effects, the No-Observed Adverse Effect Level is greater than 1500 ppm (equivalent to 91-104 and 85-147 mg DMCHA per kg body weight per day for males and females, respectively).
Executive summary:

Four groups of ten Wistar Han rats/sex were exposed to DMCHA by dietary administration at the following dose levels: 0, 150, 500 and 1500 ppm. Males received the test substance for 28 days (2 weeks prior to mating, during mating and up to necropsy). Females were exposed for 41 -54 days (2 weeks prior to mating, during mating, during post-coitum and during at least 4 days of lactation). Clinical signs, functional observations, body weights, food consumption, reproduction parameters, observations pups, clinical pathology, macroscopy, organ weights, and histopathology were evaluated. Chemical analyses of diet were conducted twice during the study to assess accuracy, homogeneity and stability. The diet was homogeneous and stable for at least 8 days at room temperature.

Decreased body weights and food consumption was reported at 1500 ppm in males and females during week 1. This was considered to be due to a palatability effect of the compound. After week 1, food consumption increased to normal values and body weight gain was normal compared to the control group. Other effects on body weights were noted in females mainly during the postcoitum phase; this decrease could not be solely explained by the food consumption in these animals. One male in the 1500 ppm dose group showed slight centrilobular hepatocellular liver hypertrophy, marked, bilateral, progressive nephropathy and slight vacuolation of the zona fasciculata of the adrenal glands together with several changes for clinical biochemistry parameters, macroscopic findings and organ weight changes. As these findings were not noted in the remaining animals of this dose group, it was not deemed to be treatment related. No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). Changes in bodyweight and food consumption at 500 ppm and 1500 ppm were not deemed to be due to DMCHA. Based on the absence of treatment related effects, the No-Observed Adverse Effect Level is greater than 1500 ppm (equivalent to 91-104 and 85-147 mg DMCHA per kg body weight per day for males and females, respectively).

According to Regulation EC No. 1272/2008 and Directive 67/548/EEC, the test substance does not require classification on the basis of the results achieved in this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
85 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline compliant studies are available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 1987 to September 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Similar to guideline study; GLP. Exposure was only two weeks.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 5 male and five female rats were exposed to 0, 5, 20 or 75 ppm nominal concentrations by whole body exposure in 1000 L inhalation chambers. The actual atmosphere concentrationswere determined analytically on three occasions and the mean exposures were calculated to be 0, 0.026, 0.104 and 0.389 mg/L. The rats were exposed to DMCHA for 6 hours per day, 5 days per week and had a total of 9 exposures on days 1-5 and 8-11.
Blood samples were collected, ophthalmic examinations completed after initial exposure, clinical signs recorded daily and bodyweights recorded regularly.
The study duration was shorter than normal for a guideline subacute study but the ocal effects of exposure to DMCHA were detectable in the study results
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Species, strain: Fischer 344 rats
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY
- Age at study initiation: 6 weeks
- Weight at study initiation: males 179±9 g, females 115±6 g
- Fasting period before study: Not applicable
- Housing:
- Diet (e.g. ad libitum): ad lib. Purina Certified Rodent Chow #5002
- Water (e.g. ad libitum): ad lib.
- Acclimation period: two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): standard laboratory environmental conditions
- Humidity (%):standard laboratory environmental conditions
- Air changes (per hr):standard laboratory environmental conditions
- Photoperiod (hrs dark / hrs light):standard laboratory environmental conditions
- Air changes: 12/hour
IN-LIFE DATES: From: To: Not stated, study commenced in December 1987
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: Not applicable for vaporised liquid
Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed in a 1000 L stainless steel and glass chamber by whole body exposure (dimensions 90x 90x 90 cm with pyrimidal top and bottom).
- Method of holding animals in test chamber:
- Source and rate of air: Compressed heated air was passed through the J-tube to volatilise the DMCHA awhich was also metered into the J-tube as a liquid. The dynamic airflow in the chamber was maintained at approximately 200L/min
- Method of conditioning air: Compressed air was heated with a flameless torch.

- Temperature, humidity, pressure in air chamber: Recorded after each 6 hour exposure and maintained to provide a chamber temperature of circa 22°C
- Air flow rate: 200L/min
- Air change rate: 12/hour
- Method of particle size determination: analytical concentration of DMCHA in the test atmosphere was determined at least 3x per exposure by gas chromatography

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography
- Samples taken from breathing zone: yes - also used to determine chamber distribution. Results indicated variation for concentration was within 16% of nominal reference values

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Measurement of chamber concentrations by gas chromatography
- nominal concentrations were calculated based on the amount of test material used and the airflow through the chamber
- Sampling times: at least 3 times per exposure period
Duration of treatment / exposure:
9 days, Exposures were conducted on test days 1-5 and 8-11
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
0 mg/m³ air (analytical)
Dose / conc.:
104 mg/m³ air (analytical)
Dose / conc.:
389 mg/m³ air (analytical)
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes
Details on study design:
Post-exposure period: Rats were subject tto necropsy on the day following the last exposure (Day 12)
- Dose selection rationale: The repeated adinistration dose concentrations were based on the results of acute investigations completed by the same investigators. Results f the acute study are also presented in the report
- Rationale for animal assignment (if not random): Random
Positive control:
Not applicable
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS AND FREQUENCY: 
- Clinical signs and mortality: daily after exposure for overt signs of toxicity or changes in demeanor. 
- Body weight: at day 1, 3, 5, 8 and 11.
- Ophthalmoscopic examination: All eyes were examined prior to the initial exposure to DMCHA (the acute study resulted in irrreversible corneal opacity) but there are no recordeds of specific ocular examintions for the remainder of the study.
- Behavioural effects: daily assessments for signs of toxicity, behavioural change or CNS effects
-Food and water consumptions were visually assessed
Terminal blood samples were obtained for haematology and clinical pathology investigations (see pathology). Urinesamples were collected prior to the last exposure period and assessed for coour, appearance and standard urinary parameters (including bilirubin, glucose, ketones, blood, ph, protein and urobilinogen, specific gravity)
Sacrifice and pathology:
Terminal blood samples were obtained from the orbital sinus.

- Haematology: Haematocrit (HCT), haemoglobin (HGB), erythrocyte (RBC), total leukocyte (WBC) and platelet (PLAT) counts. 
- Biochemistry: nitrogen (UN), alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST), alkaline phosphatase activity (AP), glucose (GLUC), total protein (TP), albumin (ALB), globulin (GLOB), cholesterol (CHOL) and triglycerides (TRIG).
- Urinalysis: immediately prior to the last exposure; bilirubin, glucose, ketones, blood, pH, protein, urobilinogen and specific gravity 

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Organ weights: brain, heart, lungs, liver, kidneys and testes 
- Macroscopic: adrenals, aorta, auditory sebaceous glands, bone (marrow), brain, cecum, cervix, coagulating glands, colon, duodenum, epididymis, oesophagus, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal glands, larynx, liver, lungs, mammary gland, mediastinal lymph node, mediastinal tissues, mesenteric tissues, nasal tissues, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus, vagina
- Microscopic: same as macroscopic except auditory sebaceous glands
Other examinations:
No further information
Statistics:
Descripive statistics for chamber concentrations, temperature, relative humidity and white blood cell counts.
Bartlett's test (equality of variance); ANOVA; Dunnett's test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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:
not examined
Details on results:
OBSERVATIONS
-no mortalities occurred 
- Clinical signs: no overt signs of toxicity or changes in demeanor were observed. 
- Body weight gain: body weights of male and female rats were statistically decreased from control values for all dose groups. Only the decreases in the 104 and 389 mg/m3 groups (males: 10% and 25% of control, respectively; females: 33% and 52% of control, respectively) were considered exposure-related. No effects on bodyweight were recorded forthe low dose concentration group

CLINICAL CHEMISTRY:
- Haematology: at 389 mg/m3 significantly decreased white blood cell counts in both sexes, but this was not considered toxicologically significant nor exposure related (due to the variability of this parameter in the historical controls and the similarity of historical control and the high concentration numbers); 
- Clinical chemistry: the following parameters were significantly deceased from control values, but were not considered to be toxicologically significant: total protein and alkaline phosphatase activity (at 26 and 389 mg/m3),  globulin (at 26 mg/m3) and albumin (389 mg/m3) 
- Urinalysis: no exposure-related effects were observed for anyof the urinary parameters evaluated 

NECROPSY FINDINGS:
- Organ weights:  at 26 mg/m3 significantly decreased absolute and relative heart weights in both sexes (not considered exposure-related, no dose response relationship);  at 389 mg/m3 increased relative kidney weights in both sexes: 2% in males and 7% in females (of questionable significance, not accompanied by changes in urinalysis, serum nitrogen or histopathologic changes in the kidney) 
- Gross pathology: no exposure related effects
- Histopathology: at 389 mg/m3 hyperplasia and hypertrophy of the nasal cavity in 3 males and 1 female
Key result
Dose descriptor:
NOAEC
Effect level:
104 mg/m³ air
Sex:
male/female
Basis for effect level:
other: based on effects on the nasal respiratory mucosa (local effect).
Dose descriptor:
NOEC
Effect level:
26 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Absence of effects on body weight.
Critical effects observed:
not specified

ANALYSES:
- Actual dose level: The nominal and analytical concentrations were in close agreement at all exposure
levels.
- Stability: acceptable
- Homogeneity: The concentration from each sample point was
within 16% of the reference point. The average of the sample
point values was within 5% of the reference point values.

In an acute inhalation investigation rats were exposed to a single 6 -hour exposure to DMCHA at concentrations of 88, 320 or 1120 ppm. All rats in the high dose group died within 2.5 hours with antecedent signs of laboured respiration and tremors. In the lower dose groups the surviving rats had lower bodyweights initially but showed good recovery. The primary effect observed was aan ophthalmic lesion - although the iris and retina were overtly unaffected, the animals developed irreversible corneal opacities. Microscopically there effects on the cornea - slight to severe opacity, inflammation and some corneal neovascularisation. The lowest dose 88 ppm resulted in only slight transient eye irrition.

In the repeated administration study, concentrations of 2, 20 or 75 ppm were used. Although the eyes were examined prior to exposure, there are no indications that the high dose concentration, 75 ppm, elicited any of the corneal effcts observed in the acute study.











Conclusions:
Local nasal epithelial irritation, evident microscopically as hyperplasia and hypertrophy, was the primary effect of toxicological significance following repeated exposure to a DMCHA atmosphere. No signs of systemic effects were noted in life at concentrations up to 104 mg/m3. Under the conditions of this study the test substance does not require classification according to Regulation EC No. 1272/20008 or Directive 67/548/EEC.
Executive summary:

In a study conducted by Nitschke and Johnson (1989), the test substance, DMCHA was tested for its ability to cause toxicity when administered via whole body inhalation. The test substance was administered to male and female Fischer 344 rats at concentrations of 0, 5, 20 or 75 ppm (equivalent to 0, 0.026, 0.104 or 0.389 mg/l) for 6 hours per day, 5 days a week for 9 exposures (test days 1 - 5 and 8 - 11). All test animals were observed daily after exposure for overt signs of toxicity or changes in demeanour. All animals were weighed on test days 1, 3, 5, 8 and 11. Animals were necropsied on the day following the last exposure and blood samples were obtained for haematologic and clinical chemistry determinations.

Slight decreases in mean body weights of male and female rats exposed to 75ppm and female rats exposed to 20ppm were observed. Slight hyperplasia and hypertrophy of the nasal respiratory mucosa was observed in four out of 10 rats exposed to 75ppm test concentration. Based on these results, the NOAEC was determined to be 104 mg/m3.

Under the conditions of this study the test substance does not require classification according to Regulation EC No. 1272/20008 or Directive 67/548/EEC.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
104 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP study

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 1987 to September 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Similar to guideline study; GLP. Exposure was only two weeks.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 5 male and five female rats were exposed to 0, 5, 20 or 75 ppm nominal concentrations by whole body exposure in 1000 L inhalation chambers. The actual atmosphere concentrationswere determined analytically on three occasions and the mean exposures were calculated to be 0, 0.026, 0.104 and 0.389 mg/L. The rats were exposed to DMCHA for 6 hours per day, 5 days per week and had a total of 9 exposures on days 1-5 and 8-11.
Blood samples were collected, ophthalmic examinations completed after initial exposure, clinical signs recorded daily and bodyweights recorded regularly.
The study duration was shorter than normal for a guideline subacute study but the ocal effects of exposure to DMCHA were detectable in the study results
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Species, strain: Fischer 344 rats
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY
- Age at study initiation: 6 weeks
- Weight at study initiation: males 179±9 g, females 115±6 g
- Fasting period before study: Not applicable
- Housing:
- Diet (e.g. ad libitum): ad lib. Purina Certified Rodent Chow #5002
- Water (e.g. ad libitum): ad lib.
- Acclimation period: two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): standard laboratory environmental conditions
- Humidity (%):standard laboratory environmental conditions
- Air changes (per hr):standard laboratory environmental conditions
- Photoperiod (hrs dark / hrs light):standard laboratory environmental conditions
- Air changes: 12/hour
IN-LIFE DATES: From: To: Not stated, study commenced in December 1987
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: Not applicable for vaporised liquid
Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed in a 1000 L stainless steel and glass chamber by whole body exposure (dimensions 90x 90x 90 cm with pyrimidal top and bottom).
- Method of holding animals in test chamber:
- Source and rate of air: Compressed heated air was passed through the J-tube to volatilise the DMCHA awhich was also metered into the J-tube as a liquid. The dynamic airflow in the chamber was maintained at approximately 200L/min
- Method of conditioning air: Compressed air was heated with a flameless torch.

- Temperature, humidity, pressure in air chamber: Recorded after each 6 hour exposure and maintained to provide a chamber temperature of circa 22°C
- Air flow rate: 200L/min
- Air change rate: 12/hour
- Method of particle size determination: analytical concentration of DMCHA in the test atmosphere was determined at least 3x per exposure by gas chromatography

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography
- Samples taken from breathing zone: yes - also used to determine chamber distribution. Results indicated variation for concentration was within 16% of nominal reference values

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Measurement of chamber concentrations by gas chromatography
- nominal concentrations were calculated based on the amount of test material used and the airflow through the chamber
- Sampling times: at least 3 times per exposure period
Duration of treatment / exposure:
9 days, Exposures were conducted on test days 1-5 and 8-11
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
0 mg/m³ air (analytical)
Dose / conc.:
104 mg/m³ air (analytical)
Dose / conc.:
389 mg/m³ air (analytical)
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes
Details on study design:
Post-exposure period: Rats were subject tto necropsy on the day following the last exposure (Day 12)
- Dose selection rationale: The repeated adinistration dose concentrations were based on the results of acute investigations completed by the same investigators. Results f the acute study are also presented in the report
- Rationale for animal assignment (if not random): Random
Positive control:
Not applicable
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS AND FREQUENCY: 
- Clinical signs and mortality: daily after exposure for overt signs of toxicity or changes in demeanor. 
- Body weight: at day 1, 3, 5, 8 and 11.
- Ophthalmoscopic examination: All eyes were examined prior to the initial exposure to DMCHA (the acute study resulted in irrreversible corneal opacity) but there are no recordeds of specific ocular examintions for the remainder of the study.
- Behavioural effects: daily assessments for signs of toxicity, behavioural change or CNS effects
-Food and water consumptions were visually assessed
Terminal blood samples were obtained for haematology and clinical pathology investigations (see pathology). Urinesamples were collected prior to the last exposure period and assessed for coour, appearance and standard urinary parameters (including bilirubin, glucose, ketones, blood, ph, protein and urobilinogen, specific gravity)
Sacrifice and pathology:
Terminal blood samples were obtained from the orbital sinus.

- Haematology: Haematocrit (HCT), haemoglobin (HGB), erythrocyte (RBC), total leukocyte (WBC) and platelet (PLAT) counts. 
- Biochemistry: nitrogen (UN), alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST), alkaline phosphatase activity (AP), glucose (GLUC), total protein (TP), albumin (ALB), globulin (GLOB), cholesterol (CHOL) and triglycerides (TRIG).
- Urinalysis: immediately prior to the last exposure; bilirubin, glucose, ketones, blood, pH, protein, urobilinogen and specific gravity 

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Organ weights: brain, heart, lungs, liver, kidneys and testes 
- Macroscopic: adrenals, aorta, auditory sebaceous glands, bone (marrow), brain, cecum, cervix, coagulating glands, colon, duodenum, epididymis, oesophagus, eyes, gross lesions, heart, ileum, jejunum, kidneys, lacrimal glands, larynx, liver, lungs, mammary gland, mediastinal lymph node, mediastinal tissues, mesenteric tissues, nasal tissues, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus, vagina
- Microscopic: same as macroscopic except auditory sebaceous glands
Other examinations:
No further information
Statistics:
Descripive statistics for chamber concentrations, temperature, relative humidity and white blood cell counts.
Bartlett's test (equality of variance); ANOVA; Dunnett's test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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:
not examined
Details on results:
OBSERVATIONS
-no mortalities occurred 
- Clinical signs: no overt signs of toxicity or changes in demeanor were observed. 
- Body weight gain: body weights of male and female rats were statistically decreased from control values for all dose groups. Only the decreases in the 104 and 389 mg/m3 groups (males: 10% and 25% of control, respectively; females: 33% and 52% of control, respectively) were considered exposure-related. No effects on bodyweight were recorded forthe low dose concentration group

CLINICAL CHEMISTRY:
- Haematology: at 389 mg/m3 significantly decreased white blood cell counts in both sexes, but this was not considered toxicologically significant nor exposure related (due to the variability of this parameter in the historical controls and the similarity of historical control and the high concentration numbers); 
- Clinical chemistry: the following parameters were significantly deceased from control values, but were not considered to be toxicologically significant: total protein and alkaline phosphatase activity (at 26 and 389 mg/m3),  globulin (at 26 mg/m3) and albumin (389 mg/m3) 
- Urinalysis: no exposure-related effects were observed for anyof the urinary parameters evaluated 

NECROPSY FINDINGS:
- Organ weights:  at 26 mg/m3 significantly decreased absolute and relative heart weights in both sexes (not considered exposure-related, no dose response relationship);  at 389 mg/m3 increased relative kidney weights in both sexes: 2% in males and 7% in females (of questionable significance, not accompanied by changes in urinalysis, serum nitrogen or histopathologic changes in the kidney) 
- Gross pathology: no exposure related effects
- Histopathology: at 389 mg/m3 hyperplasia and hypertrophy of the nasal cavity in 3 males and 1 female
Key result
Dose descriptor:
NOAEC
Effect level:
104 mg/m³ air
Sex:
male/female
Basis for effect level:
other: based on effects on the nasal respiratory mucosa (local effect).
Dose descriptor:
NOEC
Effect level:
26 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Absence of effects on body weight.
Critical effects observed:
not specified

ANALYSES:
- Actual dose level: The nominal and analytical concentrations were in close agreement at all exposure
levels.
- Stability: acceptable
- Homogeneity: The concentration from each sample point was
within 16% of the reference point. The average of the sample
point values was within 5% of the reference point values.

In an acute inhalation investigation rats were exposed to a single 6 -hour exposure to DMCHA at concentrations of 88, 320 or 1120 ppm. All rats in the high dose group died within 2.5 hours with antecedent signs of laboured respiration and tremors. In the lower dose groups the surviving rats had lower bodyweights initially but showed good recovery. The primary effect observed was aan ophthalmic lesion - although the iris and retina were overtly unaffected, the animals developed irreversible corneal opacities. Microscopically there effects on the cornea - slight to severe opacity, inflammation and some corneal neovascularisation. The lowest dose 88 ppm resulted in only slight transient eye irrition.

In the repeated administration study, concentrations of 2, 20 or 75 ppm were used. Although the eyes were examined prior to exposure, there are no indications that the high dose concentration, 75 ppm, elicited any of the corneal effcts observed in the acute study.











Conclusions:
Local nasal epithelial irritation, evident microscopically as hyperplasia and hypertrophy, was the primary effect of toxicological significance following repeated exposure to a DMCHA atmosphere. No signs of systemic effects were noted in life at concentrations up to 104 mg/m3. Under the conditions of this study the test substance does not require classification according to Regulation EC No. 1272/20008 or Directive 67/548/EEC.
Executive summary:

In a study conducted by Nitschke and Johnson (1989), the test substance, DMCHA was tested for its ability to cause toxicity when administered via whole body inhalation. The test substance was administered to male and female Fischer 344 rats at concentrations of 0, 5, 20 or 75 ppm (equivalent to 0, 0.026, 0.104 or 0.389 mg/l) for 6 hours per day, 5 days a week for 9 exposures (test days 1 - 5 and 8 - 11). All test animals were observed daily after exposure for overt signs of toxicity or changes in demeanour. All animals were weighed on test days 1, 3, 5, 8 and 11. Animals were necropsied on the day following the last exposure and blood samples were obtained for haematologic and clinical chemistry determinations.

Slight decreases in mean body weights of male and female rats exposed to 75ppm and female rats exposed to 20ppm were observed. Slight hyperplasia and hypertrophy of the nasal respiratory mucosa was observed in four out of 10 rats exposed to 75ppm test concentration. Based on these results, the NOAEC was determined to be 104 mg/m3.

Under the conditions of this study the test substance does not require classification according to Regulation EC No. 1272/20008 or Directive 67/548/EEC.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
104 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP study

Additional information

Repeated Dose Toxicity - Oral:

Four groups of ten Wistar Han rats/sex were exposed to cyclohexyldimethylamine (DMCHA) by dietary administration at the following dose levels: 0, 150, 500 and 1500 ppm. Males received the test substance for 28 days (2 weeks prior to mating, during mating and up to necropsy). Females were exposed for 41 -54 days (2 weeks prior to mating, during mating, during post-coitum and during at least 4 days of lactation). Clinical signs, functional observations, body weights, food consumption, reproduction parameters, observations pups, clinical pathology, macroscopy, organ weights, and histopathology were evaluated. Chemical analyses of diet were conducted twice during the study to assess accuracy, homogeneity and stability. The diet was homogeneous and stable for at least 8 days at room temperature.

Decreased body weights and food consumption were reported at 1500 ppm in males and females during week 1. This was considered to be due to a palatability effect of the compound. After week 1, food consumption increased to normal values and body weight gain was normal compared to the control group. Other effects on body weights were noted in females mainly during the postcoitum phase; this decrease could not be solely explained by the food consumption in these animals. One male in the 1500 ppm dose group showed slight centrilobular hepatocellular liver hypertrophy, marked, bilateral, progressive nephropathy and slight vacuolation of the zona fasciculata of the adrenal glands together with several changes for clinical biochemistry parameters, macroscopic findings and organ weight changes. As these findings were not noted in the remaining animals of this dose group, it was not deemed to be treatment related. No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). Changes in bodyweight and food consumption at 500 ppm and 1500 ppm were not deemed to be due to DMCHA. Based on the absence of treatment related effects, the No-Observed Adverse Effect Level is greater than 1500 ppm (equivalent to 91-104 and 85-147 mg DMCHA per kg body weight per day for males and females, respectively).

According to Regulation EC No. 1272/2008 and Directive 67/548/EEC, the test substance does not require classification on the basis of the results achieved in this study.

DMCHA is known to be corrosive and it was therefore administered at the highest dose level (1500 ppm, equivalent to 91-104 and 85-147 mg DMCHA per kg body weight per day for males and females, respectively)that

would not cause local effects in a 28 day study. At 1500 ppm (

equivalent to 91-104 and 85-147 mg DMCHA per kg body weight per day for males and females, respectively),

food consumption was reduced in the first week with reduced body weight. This is expected to be related to the irritating properties of the test material, or the effect on palatability. No systemic effects were reported in the study.

A 90 day repeated dose oral toxicity study (OECD 408) was conducted using gavage administration in the rat. 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.

Repeated Dose Toxicity - Dermal:

Testing cyclohexyldimethylamine (DMCHA) for repeated dermal toxicity cannot be justified on scientific grounds due to the corrosive properties of the test substance. It is not considered a suitable route of exposure

Repeated Dose Toxicity - Inhalation:

In a study conducted by Nitschke and Johnson (1989), the test substance, cyclohexyldimethylamine (DMCHA) was tested for its ability to cause toxicity when administered via whole body inhalation. The test substance was administered to male and female Fischer 344 rats at concentrations of 0, 5, 20 or 75 ppm (equivalent to 0, 0.026, 0.104 or 0.389 mg/l) for 6 hours per day, 5 days a week for 9 exposures (test days 1 - 5 and 8 - 11). All test animals were observed daily after exposure for overt signs of toxicity or changes in demeanour. All animals were weighed on test days 1, 3, 5, 8 and 11. Animals were necropsied on the day following the last exposure and blood samples were obtained for haematologic and clinical chemistry determinations.

Slight decreases in mean body weights of male and female rats exposed to 75ppm and female rats exposed to 20ppm were observed. Slight hyperplasia and hypertrophy of the nasal respiratory mucosa was observed in four out of 10 rats exposed to 75ppm test concentration. Based on these results, the NOAEC was determined to be 104 mg/m3.

Under the conditions of this study the test substance does not require classification according to Regulation EC No. 1272/20008 or Directive 67/548/EEC.

Justification for classification or non-classification

Repeated Dose Toxicity - Oral:

Based on the absence of treatment related systemic effects, the No-Observed Adverse Effect Level was determined to be greater than 1500 ppm (equivalent to 91-104 and 85-147 mg DMCHA per kg body weight per day for males and females, respectively) in the combined repeated dose/reproductive toxicity study. In the 90 -day repeated dose study, 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

According to Regulation EC No. 1272/2008 and Directive 67/548/EEC, the substance does not require classification on the basis of the results achieved in this study.

Repeated Dose Toxicity - Inhalation:

Based on the results of this study the substance does not require classification according to Regulation EC No. 1272/20008 or Directive 67/548/EEC.