Cyclohexyldimethylammonium dihydrogen phosphate

Repeated dose toxicity: oral

Administrative data

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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): DMCHA- Molecular formula (if other than submission substance): C8H17N- Molecular weight (if other than submission substance): 127g/mol- 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- Stability under test conditions: Stable- Storage condition of test material: At room temperature in the dark

Test animals

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 dayIN-LIFE DATES: From: September 30th, 2009 To: December 7th, 2009

Administration / exposure

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 for41-54 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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

150 ppm

Remarks:

nominal in diet

Doses / concentrations 3

Dose / conc.:

500 ppm

Remarks:

nominal in diet

Doses / concentrations 4

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

Examinations

Observations and examinations performed and frequency:

MORTALITY/VIABILITY: Yes - Time schedule: At least twice dailyDETAILED 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: YesWATER 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 422HAEMATOLOGY: 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 timeCLINICAL 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 acidsURINALYSIS: 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 activityOTHER: 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/groupAll 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 PCervix Clitoral gland Colon Duodenum Epididymides Eyes with optic nerve (if detectable) andHarderian gland (Female mammary gland area) Spinal cord -cervical, midthoracic, lumbarFemur including jointHeart Ileum Jejunum Kidneys (Larynx) (Lacrimal gland, exorbital)LiverLung, infused with formalin Lymph nodes - mandibular, mesenteric (Nasopharynx) Oesophagus Ovaries PancreasPeyer's patches (jejunum, ileum) if detectablePituitary glandPreputial glandProstate gland Rectum(Salivary glands - mandibular, sublingual)Sciatic nerveSeminal vesicles including coagulating glandsSkeletal muscle(Skin)Spinal cord -cervical, midthoracic, lumbarSpleen Sternum with bone marrowStomachTestes ThymusThyroid including parathyroid (if detectable)(Tongue)TracheaUrinary bladderUterusVaginaAll gross lesionsAll remaining animals and females which failed to deliver 2:CervixClitoral gland Coagulation glandEpididymidesOvariesPreputial gland Identification marks: not processedProstate gland Seminal vesiclesTestes UterusVaginaAll gross lesionsHISTOPATHOLOGY: YesAll 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 parathyroidAll 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 displaydifferent test statistics values.No statistical analysis was performed on histopathology findings.

Results and discussion

Results of examinations

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 MORTALITYNo 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 GAINBody 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 animalsduring 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.HAEMATOLOGYNo changes in haematological parameters were reported. Changes that occurred were considered to be of no toxicological relevance.CLINICAL CHEMISTRYNo 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 WEIGHTSThe following statistically significant changes in organ weights distinguished treated from controlanimals:- 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 ppmIn 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 PATHOLOGYNo 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-NEOPLASTICNo treatment related microscopic findings were reported. One male at 1500 ppm showed slight centrilobularhepatocellular 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 oradrenal gland toxicityHISTOPATHOLOGY: 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 FINDINGSNo 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.

Effect levels

1

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.

2

Key result

Dose descriptor:

NOAEL

Remarks:

corrected for mean test article intake

Effect level:

>= 85 - <= 147 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: NOAEL = highest dose tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

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)	236^a	-	225^a	216 ± 2.8^b
Day 14 (Week 2)	235^a	-	-	218^a
Day 22 (week 4)	234^a	-	-	229^a
Day 29 (week 5)	235^a	-	-	216^a

^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)	17^a	-	8^a	5 ± 1.4^b
Day 14 (Week 2)	16^a	-	-	6^a
Day 22 (week 4)	16^a	-	-	11^a
Day 29 (week 5)	16^a	-	-	5^a

^aonly one female was weighed

^btwo females were weighed

Applicant's summary and conclusion

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.