Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 3 JAN 2007 to 22 FEB 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 422)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
but according to the study authors none of these deviations affected the study's integrity (for details please see "Any other information on materials and methods incl tables").
GLP compliance:
yes
Remarks:
according to OECD GLP Guidelines (1997)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dytek® DCH-99, 1,2-Diaminocyclohexane (DCH)
- Substance type: clear light-yellow
- Physical state: liquid
- Analytical purity: 99.5%
- Composition of test material, percentage of components:
99.55% 1,2-Diaminocyclohexane (CAS 694-83-7)
0.24% 2-Aminomethylcyclopentylamine
(CAS 21544-02-5)
0.08% Hexamethyleneimine (CAS 111-49-9)
0.00% Hexamethylenediamine (CAS 124-09-4)
0.07% 2-Methy11,5-pentamethylenediamine
(CAS 15520-10-2)
0.06% Water
- Stability under test conditions: test material in vehicle is stable for at least 5 hours when stored at room temperature
- Storage condition of test material: at ambient temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: (P) approx. 10 wks
- Weight at study initiation: (P) Males mean: 336.75 g; Females mean: 229 g
- Housing:
#Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
#Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
#Post-mating: Males were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). Females were individually housed in Macrolon cages (MIII type, height 18 cm).
#Lactation: Offspring was kept with the dam until termination.
General Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During overnight activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during overnight activity monitoring.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest,Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-23.3
- Humidity (%): 29-87%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8 JAN 2007 To: 22 FEB 2007

FURTHER DETAILS:
Animals were housed in Room 16 until 09 January 2007 and in Room 3 from 09 January 2007 (2nd day of substance treatment) onwards.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(Milli-U from Millipore Corporation, Bedford, USA)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the test substance.
- Rationale for vehicle: Based an trial formulations performed at NOTOX.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Group Analysis (type of sample)
1 acc (M)
2 acc + hom + stabt.0 (TMB), stabt=5, RT (S)
3 acc (M)
4 acc + hom + stabt.0 (TMB), stabt.5, RT (S)

Duplicate samples were analysed
acc=accuracy, hom=homogeneity, stab=stability (hours), T=top, M=middle, B=bottom position of container
S=stability sample taken at middle position of container
Duration of treatment / exposure:
(P) Males: Overall males were exposed for 31 days (two weeks prior to mating, during mating and up to termination)
(P) Females: Overall females were exposed for 42 to 45 days (2 weeks prior to mating, during mating, during post-coitum, and at least 3 days of lactation)
Frequency of treatment:
Once daily for 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150, and 500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:based on findings from dose-range finding study
- dose volume: 20 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily; Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard
arena. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

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

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes (see functional observation battery)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (overnight, with a maximum of 20 hours)
- How many animals: 5 males and 5 females per group
- Parameters checked: 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: immediately prior to scheduled post mortem examination
- Animals fasted: Yes
- How many animals: 5 males and 5 females per group
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic, Phosphate

URINALYSIS: No

OTHER: Functional Observations
The following tests were performed in 5 males and 5 females, randomly selected from each group:
# hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent).
# motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
During the motor activity test, males were caged individually and females were caged with their offspring.
The assigned males were tested during week 4 of treatment and the assigned females were tested during lactation (all before blood sampling).
In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 minutes.

OTHER:
Observations on females and litters, calculation of reproduction parameters (male and females) are reported in section 7.8.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (gross necropsy consisted of external and internal examinations including the cranial, thoracic, and abdominal tissues and organs, with special attention being paid to the reproductive organs)
HISTOPATHOLOGY: Yes (various organs and tissues from at least 5 selected animals per sex per group were collected)
histopathologic examinations were performed on:
- The preserved organs and tissues of the selected animals of Groups 1(control) and 4 (high dose).
- gender specific investigation (e.g. The additional slides of the testes of the selected 5 males/group of Groups 1 and 4 to examine staging of spermatogenesis, further details see above)
- All gross lesions of all animals (all dose groups)

On detection of possible treatment-related changes in the organs of any animal in the high dose group, histological examination was extended to lungs, thymus, liver, kidneys (all males and females) and adrenals (males only) of five selected animals of Groups 2 and 3.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
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 (many-to-one t-test) based on a pooled variance estimate were applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data could not assumed to follow a normal distribution.
• The Fisher-exact test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. 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 sam printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no animal died during the study, 2 females were killed due to total litter loss; high dose group: Slight to moderate salivation was noted in all males and females treated; incidentally rales were noted in two males
Mortality:
mortality observed, treatment-related
Description (incidence):
no animal died during the study, 2 females were killed due to total litter loss; high dose group: Slight to moderate salivation was noted in all males and females treated; incidentally rales were noted in two males
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
high dose group: reduced body weights and body weight gains of male and female animals
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
high dose group: food consumption was reduced in females during lactation
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
high dose group: decrease in relative number of eosinophils
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
high dose group mainly in males: high alanine aminotransferase, aspartate aminotransferase (also noted in females at 500 mg/kg) and alkaline phosphatase activities and high cholesterol levels
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
high dose group: increase in activity at the low sensor in the motor activity test
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
high dose goup males: increased liver/body weight ratios
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
high dose group: findings in liver and lung
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- No treatment related mortality occurred during the study period.
- Two females were sacrificed before the end of the study period. One animal treated at 50 mg/kg (Female 60) was sacrificed as the animal was cannibalizing the pups on Day 1 of lactation and one female at 500 mg/kg (Female 79) was sacrificed due to total litter loss on Day 2 of lactation.
This animal had five pups, three pups were found dead on Day 1 of lactation, and two other pups were found dead on Day 2 of lactation.

- high dose group: Slight to moderate salivation was noted in all males and females treated; incidentally rales were noted in two males (Males 37 and 38) and piloerection was noted in one female (Female 79) at the end of treatment.
- all dose groups except control: yellow discolouration of the urine was noted in all animals

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Slightly reduced body weight and body weight gain was noted in males treated at 500 mg/kg during the complete study period (not always statistically significant). Reduced body weight and body weight gain was also noted in females treated at 500 mg/kg on Days 14 to 20 post-coitum and during lactation (not always statistically significant).

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Food consumption before or after allowance for body weight was reduced during lactation in females treated at 500 mg/kg (statistically not significant). Other changes in food consumption were considered to be of no toxicological relevance.

HAEMATOLOGY
- A decrease in relative number of eosinophils was noted in males and females at 500 mg/kg.
- Furthermore, a slight increase in red blood cell distribution width was noted in males at 500 mg/kg. However as no corroborative findings were noted in related parameters this finding was considered to be of no toxicological relevance.
- Other findings achieving statistical significance (increased APTT in males at 50 and 150 mg/kg) were considered to be of no toxicological relevance in absence of a dose response relationship.

CLINICAL CHEMISTRY
- Treatment related effects were mainly noted in males treated at 500 mg/kg. These findings comprised high alanine aminotransferase, aspartate aminotransferase (also noted in females at 500 mg/kg) and alkaline phosphatase activities and high cholesterol levels. Cholesterol levels were also increased in males treated at 150 mg/kg, but to a lesser extent.
- Furthermore, calcium levels were increased in males and females at 500 mg/kg.
- Other findings achieving statistical significance (decreased potassium levels in males at 50 mg/kg and increased albumin in females at 150 mg/kg) were considered to be of no toxicological relevance in absence of a dose response relationship.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- high dose group: Liver/body weight ratlos were increased with statistical significance for males, which correlated with the findings at macroscopic and microscopic examination; decreased epididymides weight in males
- high dose group: Decreased thymus weight and thymus/body weight ratlos were noted in males and females and correlated in some animals with the atrophy noted at microscopic examination. Furthermore, increased kidney weight and kidney/body weight ratlos were noted in females, which correlated in some animals with the observed basophilia. However, as these microscopic findings were not considered to be related to treatment, the
toxicological relevance of these organ weight changes remains unclear.
- Other changes in organ weight comprised increased heart weight in males at 50, 150 and 500 mg/kg and females at 500 mg/kg (not always statistically significant), increased relative brain weight in males at 500 mg/kg and increased adrenal weight in females at 150 and 500 mg/kg.
- Other changes (increased liver weight in females at 50 mg/kg, decreased testes weight in males at 50 mg/kg) were considered to be of no toxicological relevance in absence of a dose response relationship.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- high dose group: pale discolouration of the liver was noted in five males; many grey-white foci were found an the lungs of 4 females
- One female at 50 mg/kg (Female 60) showed reddish contents in the stomach. This animal was sacrificed due to cannibalism of the pups.
- One female at 50 mg/kg (Female 51) showed an enlarged cervix and uterus. The uterus was filled with fluid. These were signs of pseudo pregnancy and were considered to be unrelated to treatment.
- Incidental findings among control and treated animals are occasionally seen among rats used in these types of study and in the absence of a dose response relationship they were considered changes of no toxicological significance.

HISTOPATHOLOGY
- lungs
# alveolar macrophage foci and lymphocytic alveolar inflammation
(In the Jungs, alveolar macrophage foci were noted at a minimal degree in one group 3 (150 mg/kg/day) and at a slight degree in one group 4 male animals. In females this finding was present at a slight degree of severity in one Group 1 (0 mg/kg/day, control) and at slight or moderate severity in five group 4 animals which was significantly increased (p <0.05). In the same organ lymphocytic alveolar inflammation was recorded at a slight degree in one group 1, minimal in one group 3 and also at a minimal degree in four group 4 males. In females, one animal in each of Groups 1, 2 (50 mg/kg/day) and 3 recorded this finding at a minimal degree and in Group 4, five animals at minimal to moderate degree. Again this was significantly
increased in Group 4 females (p <0.05) and there was a positive trend (p <0.05) in males. These findings correlated to the gray-white foci observed in females at 500 mg/kg. At macroscopy, these findings were not observed in males.)
- liver
#In the liver, hepatocellular vacuolation at a minimal or slight degree was seen in four Group 4 males and in three group 4 females. This incidence did not reach statistical significance in either sex, however there was a positive trend (p at least <0.05) for both sexes. This finding was the microscopic correlate to the pale discolouration in this organ noted at necropsy.
-Kidneys
Corticomedullary tubular basophilia at a minimal or slight degree was recorded in the kidneys of males: two in Group 1, one in Group 3 and two in Group 4; in females: one in Group 1 and three in Groups 2 and 4. This was neither significantly increased or positive in trend for either sex.
-Adrenal glands:
In the adrenal glands of males vacuolation in the zona fasiculata was seen at a minimal degree in two group one, two group 2, slight degree in one group 3 and at minimal or slight degree in four group 4 animals. This slight increase in group 4 was not significant, however again there was a positive trend (p <0.05).
-thymus
Lymphoid atrophy — involution of the thymus was recorded in males: at minimal degree one group 1, slight in one group 3 and slight in two group 4 animals; in females: at minimal to moderate in four group 1, minimal in one group 3 and slight or moderate in three group 4 animals. This was neither significantly increased or positive in trend for either sex.

OTHER FINDINGS
- Functional observations: Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The motor activity test showed an increase in activity at the low sensor for females at 500 mg/kg. No other effects were noted in the motor activity test.

REPRODUCTIVE FUNCTION and PERFORMANCE (PARENTAL ANIMALS) and DEVELOMENTAL EFFECTS
Details are presented in section 7.8.

Effect levels

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: various findings in high dose group (e.g. reduced body weights and bw gain, haematological and clinical biochemistry findings, macroscopic and microscopic findings in liver, lung and adrenal glands)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In this OECD 422 study in rats the orally administered test material induced significant changes only in the highest dose tested; i.e. changes in clinical appearance (slight salivation), functional observations (slight hyperactivity), body weights and food consumption (decreased), clinical laboratory investigations (e.g. decreased eosinophils, ALT and AST activity increased), macroscopic and microscopic examinations (liver, lung and adrenal glands), which correlated with changes in organ weights. From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for the test item of 150 mg/kg bw/day was established.
Executive summary:

The test material was administered by daily oral gavage to male and female Wistar rats at dose levels of 0, 50, 150 and 500 mg/kg/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 31 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 3 days of lactation (for 42 to 45 days). Formulation analysis showed that the formulations were prepared accurately, were homogeneous and were stable for at least 5 hours at room temperature.

No treatment related mortality occurred during the study period.

Slight to moderate salivation was noted in all males and females treated at 500 mg/kg. Furthermore, incidentally rales were noted in two males at 500 mg/kg and piloerection was noted in one female at 500 mg/kg at the end of treatment.

Yellow discolouration of the urine was noted in all animals of Groups 2, 3 and 4 in a dose dependant manner. This could be due to excretion of the test compound or a metabolite in the urine. Without corroborative findings for clinical biochemistry parameters and as no macroscopic or microscopic abnormalities of the kidneys were observed, this finding was not considered toxicological relevant.

At 500 mg/kg, reduced body weight gain was noted in males during the treatment period and in females on Days 14 to 20 post-coitum and during lactation (not always statistically significant). Furthermore, at 500 mg/kg food consumption before or after allowance for body weight was reduced during lactation in females (statistically not significant).

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals, The motor activity test showed an increase in activity at the low sensor for females at 500 mg/kg, which might be due to hyperactivity of the dam and/or pups.

At 500 mg/kg, a treatment related decrease in eosinophils was noted in males and females. Furthermore, treatment related effects were noted in clinical biochemistry (mainly in males). These findings comprised high alanine aminotransferase, aspartate aminotransferase activities (also noted in females at 500 mg/kg) and alkaline phosphatase activities and high cholesterol levels. Cholesterol levels were also increased in males treated at 150 mg/kg, but to a lesser extent. These findings at 500 mg/kg correlated with the macroscopic or microscopic effects on the liver, e.g. pale discolouration, increased liver/body weight ratios and hepatocellular vacuolation of the liver at a minimal or slight degree. In addition, calcium levels were increased in males and females at 500 mg/kg. Besides microscopic changes in the liver, minor treatment related morphological alterations were noted in the lungs and adrenal glands: In the lungs, alveolar macrophage foci were increased in incidence and severity to moderate in females at 500 mg/kg. In the same organ lymphocytic alveolar inflammation was slightly increased in incidence in males and in incidence and severity to moderate in females. These findings correlated with the grey-white foci observed in females at 500 mg/kg. In the adrenal glands of males vacuolation in the zona fasiculata at minor degrees of severity was sbightly increased in incidence at 500 mg/kg which was not statistically significant. However there was a positive trend. The findings in liver, lung and adrenal glands were chiefly minor in nature and may be regarded as either slight increases in spontaneously occurring conditions or adaptive. As such they were considered to be indicators of slight toxicity to the test-item.

The organ weight changes in thymus and kidney correlated with the microscopic findings in these organs, e.g. atrophy and basophilia respectively. No corroborative findings were noted for the changes in weight of the heart, epididymides and brain. These changes were mild in nature and in absence of corroborative findings or a clear dose response relationship, the toxicological relevance of these changes remains unclear.

Based on these findings, the No Observed Adverse Effect Level (NOAEL) of this study was established at 150 mg/kg body weight/day.