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Toxicological information

Toxicity to reproduction

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Endpoint:
toxicity to reproduction
Remarks:
other: Repproductive toxicity/fertility and developmental toxicity Screening test
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 GLP compliant.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
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.

Reason / purpose for cross-reference:
reference to same study
Reference
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
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD guideline 422
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes
Limit test:
no
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 day


IN-LIFE DATES: From: September 30th, 2009 To: December 7th, 2009
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 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.
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.
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

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. 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.

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 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:
Cervix
Clitoral gland
Coagulation gland
Epididymides
Ovaries
Preputial gland
Identification marks: not processed
Prostate gland
Seminal vesicles
Testes
Uterus
Vagina
All gross lesions
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: No data
Examinations 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
Details on maternal toxic effects:
Maternal toxic effects:yes

Details 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.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 85 - <= 147 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details 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.
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 ppm
Based on:
test mat.
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
Key result
Abnormalities:
no effects observed
Key result
Developmental 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 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.

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

Constituent 1
Chemical structure
Reference substance name:
Cyclohexyldimethylamine
EC Number:
202-715-5
EC Name:
Cyclohexyldimethylamine
Cas Number:
98-94-2
Molecular formula:
C8H17N
IUPAC Name:
N,N-dimethylcyclohexanamine
Constituent 2
Reference substance name:
N,N-dimethylcyclohexylamine
IUPAC Name:
N,N-dimethylcyclohexylamine
Constituent 3
Reference substance name:
DMCHA
IUPAC Name:
DMCHA
Details on test material:
- Name of test material (as cited in study report): DMCHA
- Physical state: Liquid
- Purity test date: 99.4% w/w
- Lot/batch No.: 570071
- Expiration date of the lot/batch: 20 May 2011
- Molecular Weight: 127g/mol
- Molecular Formula: C8H17N
- Storage: At room temperature in the dark
- Stability: Stable

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). 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 day


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

Administration / exposure

Route of administration:
oral: feed
Type of inhalation exposure (if applicable):
other: Not applicable
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 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

Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabited with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates). Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the oestrus cycle and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 13 days was allowed for mating. After 13 days of mating, females who had not shown
evidence of mating were separated from their males.
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.
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 14 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Remarks:
nominal in diet
Dose / conc.:
150 ppm (nominal)
Remarks:
nominal in diet
Dose / conc.:
500 ppm (nominal)
Remarks:
nominal in diet
Dose / conc.:
1 500 ppm (nominal)
Remarks:
nominal in diet
No. of animals per sex per dose:
5 animals/sex/dose
Control animals:
yes, plain diet
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.

Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
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. 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
- 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:
- Animals fasted: Yes, animals were fasted overnight
- 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
Oestrous cyclicity (parental animals):
No specific information is available on the oestrus cycle
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymides weight, examination of staging of spermatogenesis.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, body weights,
early postnatal pup development (mortality and clinical signs)

GROSS EXAMINATION OF DEAD PUPS:
Gross external necropsy. 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.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: All surviving females which delivered were killed on lactation Days 5-7. All surviving females which failed to deliver were killed
post-coitum on Day 26 (female no. 49, with evidence of mating) and 21 days after the last day of the mating period (female nos. 45 and 74, without evidence of mating).

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:
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.
Postmortem examinations (offspring):
SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic examination).

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.
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.
Reproductive indices:
The following parameters were analysed:
percentage mating males, percentage mating females, fertility index males, fertility index females, conception rate, gestation index, duration of gestation
Offspring viability indices:
The following parameters were analysed:
percentage live males at first litter check, percentage live females at first litter check, percentage of post-natal loss days (0-4) of lactation, viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
see results below
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see results below
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
see results below
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
see results below
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: see results below

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
see results below

Details on results (P0)

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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
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. Percentage mating, fertility index, conception rate, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. No toxicologically significant effects on reproductive parameters were noted.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No changes were deemed to be due to DMCHA
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.

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 (PARENTAL ANIMALS)
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 historical control range among rats of this age and strain.

HISTOPATHOLOGY (PARENTAL ANIMALS)
One female in the 150 ppm dose group displayed a mammary gland adenocarcinoma. However, this is a common finding in this strain of rats of this age.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 500 ppm
Sex:
male/female
Basis for effect level:
other: No systemic toxicity was observed at 1500 ppm (the highest dose level tested)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
see results below
Mortality / viability:
no mortality observed
Description (incidence and severity):
see results below
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see results below
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
see results below
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
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) were unaffected by treatment.

CLINICAL SIGNS (OFFSPRING)
Incidental clinical symptoms op pups consisted of small size, blue spot on the nose, and cold appearance.

BODY WEIGHT (OFFSPRING)
Body weights of pups were slightly decreased at 500 and 1500 ppm on Days 1 and 4 of lactation.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination revealed autolysis for one pup that was found dead at first litter check, and small size was noted for a few pups. No relationship with treatment was established for these observations and they were considered to be of no toxicological significance.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not measured/tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Reproduction Data F0-generation.                Group 1    Group 2    Group 3     Group 4

                                                        Control 150 PPM       500 PPM    1500 PPM

Paired Males                                          10          10               10                10

Mated Males                                           9           10               10                 9

Males generating a pregnancy                      8           10               10                 9

Paired Females                                       10          10               10                10

Mated Females                                         9           10              10                  9

Non-pregnant Females                                 1            0                 0                 0

Pregnant Females                                      8           10               10                  9

Number of litters with living pups on Day 1          8           10               10                  9

 

 

 

Group 1
control

Group 2
150 ppm

Group 3
500 ppm

Group 4
1500 ppm

Percentage mating (Males) 

(Males mated / Males paired) * 100

90.0

100

100

90.0

Fertility index (Males)

(Males generating a pregnancy / Males paired) * 100

80.0

100

100

90.0

Percentage mating (Females)

(Females mated / Females paired) * 100

90.0

100

100

90.0

Fertility index (Females)

(Females achieving a pregnancy / Females paired) * 100

80.0

100

100

90.0

Conception rate

(Females achieving a pregnancy / Females mated) * 100

88.9

100

100

100

Gestation index

(Number of females with living pups / Number of females

pregnant) * 100

100

100

100

100

 

Applicant's summary and conclusion

Conclusions:
Further to the administration of DMCHA, changes in in body weights were reported. However, these changes were within the historical control and were therefore not deemed to be treatment related. No changes for reproductive parameters were revealed. On this basis, the the following No Observed Adverse Effect Levels (NOAEL) were derived parental NOAEL at least 1500 ppm (equivalent to 91-104 mg DMCHA/kg bw in males and 85-147 mg DMCHA/kg bw in females) , reproductive NOAEL at least 1500 ppm(equivalent to 91-104 mg DMCHA/kg bw in males and 85-147 mg DMCHA/kg bw in females) and developmental NOAEL at least 1500 ppm (equivalent to 91-104 mg DMCHA/kg bw in males and 85-147 mg DMCHA/kg bw in females)
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, observation of 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 change was attributed 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 parameters studied (i.e. clinical appearance, functional observations, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). No parental effects were observed at 150 ppm. No reproductive effects were observed at any dose level. Decrease in body weights in pups were reported in the 500 and 1500 ppm dose group, which are at the same dose levels as effects on maternal body weight were noted. No other developmental effects were observed at 500 and 1500 ppm. No treatment-related changes were noted in any of the developmental parameters studied (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. 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.  

Based on these results, the test substance does not require classification according to Regulation EC No. 1272/2008 or Directive 67/548/EEC.