Cyclohexyldimethylammonium dihydrogen phosphate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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 is in compliance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 422

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:

CAS #98-94-2, Air Products and Chemicals, Lot #AW4 10736M4

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

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). Animals were housed in groups of 5 animals/sex/cage during the pre-mating period. During the mating period, females were caged together with males on a one-to-one-basis in Macrolon (type MIII) cages. Post-mating, males were housed in their home cage (MIV type) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type). Sterilised sawdust was used as bedding material and paper as cage enrichment.- 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 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 substanceDIET 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.

Details on mating procedure:

- Impregnation procedure: cohoused- If cohoused:- M/F ratio per cage:one male with one female- Length of cohabitation:maximum of 13 days- After 13 days of unsuccessful pairing replacement of first male by another male with proven fertility.- Proof of pregnancy: evidence of sperm in the vaginal lavage, by staging of the oestrus cycle and/or by the appearance of an intravaginal copulatory plug

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.

Duration of test:

Males were exposed for 28 days. Females were exposed for 41 - 54 days.

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 consumptionwith slight receovery was noted. On this basis, 1500 ppm was selected as the highest dose to be tested.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No dataMORTALITY/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. 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.POST-MORTEM EXAMINATIONS: Yes / No / No data- Sacrifice on gestation day Termination was scheduled for female which delivered on days 5-7 of lactation, feamles 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)- Organs examined: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 OvariesPancreasPeyer's patches (jejunum, ileum) if detectablePituitary glandPreputial glandProstate glandRectum(Salivary glands - mandibular, sublingual)Sciatic nerveSeminal vesicles including coagulating glandsSkeletal muscle(Skin)Spinal cord -cervical, midthoracic, lumbarSpleenSternum with bone marrowStomachTestes ThymusThyroid including parathyroid (if detectable)(Tongue)TracheaUrinary bladderUterusVaginaAll gross lesionsAll remaining animals and females which failed to deliver:CervixClitoral gland Coagulation glandEpididymidesOvariesPreputial gland Identification marks: not processedProstate glandSeminal vesiclesTestes UterusVaginaAll gross lesions

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No dataExaminations included:- Gravid uterus weight: No since the study is a screening reproductive/developmental study, it does not cover the developmental toxicity only.- Number of corpora lutea: Yes - Number of implantations: Yes - Number of early resorptions: No - Number of late resorptions: No

Fetal examinations:

- External examinations: The study was not terminated during gestation. However, it provides information on the pups and external examination was carried out. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.- Soft tissue examinations: No- Skeletal examinations: No - Head examinations: No

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.

Indices:

Male fertility index.Female fertility index.Gestation index.Viability index.

Historical control data:

Yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yesDetails on maternal toxic effects:At 1500 ppm, parental effects consisted of decreased body weights and food consumption for males and females. These effects comprised an apparent decrease in food consumption in the first week of treatment, followed by a decrease in body weights in these males and females. This was considered to be due to a palatability effect of the compound. After this first week, 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 treated at 1500 ppm 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 considered to have occurred by chance and not due to DMCHA treatment.At 500 ppm, parental effects consisted of decreased body weights for females, mainly during the post-coitum phase. No treatment-related changes were noted in any of the remaining parental parameters investigated in this study.No reproductive effects were observed at any dose level.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yesDetails on embryotoxic / teratogenic effects:Developmental effects consisted of decreased body weights of pups at 500 and 1500 ppm. No other developmental effects were observed at 500 and 1500 ppm. No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 500 ppm

Based on:

test mat.

Remarks:

dams were dosed

Sex:

male/female

Basis for effect level:

other: Changes in bodyweight and food consumption at 500 and 1500 ppm were considered to be slight in nature and were not considered to be adverse. Therefore, there were no effects observed.

Remarks on result:

not measured/tested

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental 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 for maternal toxicity is greater than 1500 ppm, equivalent to 85 - 147mg/kg body weight per day.

Executive summary:

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 but were considered to be a palatable effect. Developmental effects consisted of decreased body weights of pups at 500 and 1500 ppm, which is at the same dose levels as effects on maternal body weight were noted and therefore are considered to be secondary to maternal toxicity. No other effects were observed at 500 and 1500 ppm. No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index, duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and early postnatal pup development (mortality, clinical signs and external macroscopy)). No developmental effects were observed at 150 ppm. However, the study was not designed to look at abnormalities in foetuses. Based on the absence of treatment related effects, the No-Observed Adverse Effect Level for maternal toxicity is greater than 1500 ppm, equivalent to 85 - 147mg/kg body weight per day.   Based on these results, the test substance does not require classification according to Regulation EC No. 1272/2008 or Directive 67/548/EEC.