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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 22, 2011 - August 3, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in compliance with GLP and according to OECD Guideline 422 and EPA OPPTS 870.36500.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
1564279-60-4
IUPAC Name:
1564279-60-4
Constituent 2
Reference substance name:
4,4'-methylenebis(N-secbutylcyclohexanamine)
IUPAC Name:
4,4'-methylenebis(N-secbutylcyclohexanamine)
Details on test material:
- Name of test material (as cited in study report): MTDID 25575
- Physical state: Clear colourless liquid
- Analytical purity: 98.69 Wt%
- Batch No.: 30103 260308 031
- Expiration date of the batch: 30 March 2012
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
other: Crl:WI (Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deautschland, Sulzfeld, Germany
- Age at study initiation: Approximately 15 weeks
- Weight at study initiation: Males: 357-413 g; Females: 214-253 g
- Housing: In groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). This was also applicable for recovery animals throughout the complete study period. During the mating the main females were caged together with main males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18cm). Post-mating the main males were housed in their home cages with a maximum of 5 animals/cage. Main females were individually housed in Macrolon plastic cages (MIII type, height 18cm). Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18cm).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At leas 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): A temperature of 21.0 ± 3.0 °C (actual range (REES): 19.0 - 20.8 °C)
- Humidity (%): Relative humidity of 40-70% (actual range (REES): 48-86%).
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours darkness and 12 hours artificial fluorescent light per day.

IN-LIFE DATES: From: 22 April 2011 To: 03 August 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036 (Merck, Darmstadt, Germany)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and test substance.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX and on information provided by the sponsor.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatments phase, according to a validated method (NOTOX Project 495946). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
Duration of treatment / exposure:
Main males and Recovery males were exposed for 29 days, including at least 2 weeks of exposure prior to mating and during the mating period
for Main males. The Main females were exposed for 42-45 days prior to mating, during mating, during the post coitum and lactation periods, and
up to the day prior to scheduled necropsy. Females 69, (Group 1) 81, 85 (Group 3) and 92 (Group 4) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1 , 3 and 10 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Main males and main females: 10 animals/sex/dose. Recovery (0 and 10 mg/kg bw/day): 10 males/dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dese levels were based on the dose range finding study (NOTOX Project 495982).
Positive control:
No

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, at least immediately after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed 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:
- Time schedule: Weekly, except for males and females which were housed together for mating.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period (from selected 5 animals/sex/group) and end of 14-day and 28-day recovery period.
- Anaesthetic used for blood collection: Yes (isofurane, Abbott B.V., Hoofddorp, The Netherlands)
- Animals fasted: Yes (max 20 hours)
- How many animals: 5 animals/sex/group and from all recovery males
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period (from selected 5 animals/sex/group) and end of 14-day and 28-day recovery period.
- Animals fasted: Yes (max 20 hours)
- How many animals: 5 animals/sex/group and from all recovery males
- Parameters checked in table [No.?] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected Man and Recovery males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling).
- Dose groups that were examined: All dose groups (males/females) and recovery groups (males).
- Battery of functions tested: sensory activity, grip strength and motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see Table 1 (All macroscopic findings) which have been added as a background material.
HISTOPATHOLOGY: Yes, see Table 3 (Microscopic findings) which have been added as a backgroung material.
Statistics:
The following statistical methods were used to analyze 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 was 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 if the data could not be assumed to
follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See sectioin "Details on results".
Food consumption and compound intake (if feeding study):
no effects observed
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 sectioin "Details on results".
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See sectioin "Details on results".
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
See sectioin "Details on results".
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See sectioin "Details on results".
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See sectioin "Details on results".
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See sectioin "Details on results".
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- No clinical signs of toxicity were noted during the observaton period.
- No treatment-related mortality occurred during the study period.

BODY WEIGHT AND WEIGHT GAIN
- Males at 10 mg/kg showed a slight, but statistically significant, lower mean body weight gain throughout the treatment period (for absolute body w eights from Day 22 onwards) and during the first two weeks of the recovery period (including absolute body weights). For females at 10 mg/kg, sli ghtly reduced mean weight gain/slight weight loss was noted during the first two weeks of treatment. During the post-coitum and lactation period, mean body weights remained slightly lower than control means from Day 7 onwards (body weight gain was also statistically significant lower on Day 11), being statistically significant on most occasions.

- No toxicological significance was ascribed to the statistically significant lower mean body weight of females at 1 mg/kg on Day 20 of the post-coit um phase, since a dose-related trend was absent. On other occasions, body weights and body weight gain of animals at 1 and 3 mg/kg remained in the same range as controls over the treatment period.

HAEMATOLOGY
- The following statistically significant changes in haematology parameters distinguished males at 10 mg/kg from control animals at the end of trea tment: higher relative neutrophil counts, lower relative lymphocyte counts, higher relative monocyte counts, higher platelet counts and higher prot hrombin time (PT). These changes had resolved during the 14-day recovery period.
- Other statistically significant changes in haematology parameters were considered to be without toxicological relevance.

CLINICAL CHEMISTRY
- The following statistically significant changes in clinical biochemistry parameters distinguished animals at 10 mg/kg from control animals: higher a lanine aminotransferase activity (ALAT) in males, higher aspartate aminotransferase activity (ASAT) in males and females, lower total protein levels in males and females, lower albumin levels in males and lower cholesterol levels in males and females and lower potassium levels in females. Followi ng a 14-day recovery period, aspartate aminotransferase activity remained slightly higher in males at 10 mg/kg, along with lower total protein level s. These changes had resolved during the 28-day recovery period.

NEUROBEHAVIOUR
- Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
- Kidney weight and kidney to body weight ratio of males and females at 10 mg/kg was higher than controls at the end of the treatment period (not s tatistically significant for kidney weight of females). Other statistically significant organ weight changes were considered to be without toxicological
relevance. These included a higher adrenal and testes weight, higher kidney, adrenal, testes and epididymides to body weight ratio of males at the e nd of the 14- or 28-day recovery period (findings absent at the end of treatment), respectively, higher adrenal and epididymides to body weight rat io of males at 10 mg/kg at the end of the treatment period (absolute weights were similar to control levels)

GROSS PATHOLOGY
- Necropsy did not reveal any toxicologically relevant alterations.

HISTOPATHOLOGY: NON-NEOPLASTIC
- There were treatment related microscopic findings in adrenal glands, kidneys (males only), liver, lungs, mesenteric lymph node, prostate gland, ske letal muscle and urinary bladder.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based on urothelial hyperplasia of the urinary bladder.
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on urothelial hyperplasia of the urinary bladder.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD Guideline 422 was conducted with MTDID 35575 (trade name Clearlink 1000) in order to evaluate its potential toxicity in these endpoints. Study was conducted in compliance with GLP
Test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 1, 3 and 10 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating,
during post-coitum, and at least 4 days of lactation (for 42-45 days). An additional 5 males/group in Groups 1 and 4 underwent a recovery period for 14 or 28 days.

Parental results:
Treatment with MTDID 25575 up to 10 mg/kg was well tolerated by the animals during the exposure period. Except for slightly lower body weights for males and females during the treatment/post-coitum/ lactation period, there were no in-life findings indicative of toxicity.

Haematology changes occurred in males at 10 mg/kg and included higher relative neutrophil and monocyte counts, lower relative lymphocyte counts, higher platelet counts, and higher prothrombin time. These changes has resolved during the recovery period. Clinical biochemistry changes at 10 mg/kg in males and/or females included higher alanine and aspartate aminotransferase activity, lower total protein, albumin, cholesterol and potassium levels. Following a 14-day recovery period, aspartate aminotransferase activity remained slightly higher in males at 10 mg/kg, along

There were morphological alterations in lungs (alveolar macrophage foci were noted at increased severity up to moderate degree in group 3 (3 mg/kg/day) and 4 (10 mg/kg/day) males and in group 4 females), liver (diffuse midzonal/centrilobular hypertrophy was recorded at minor (minimal or slight) degrees of severity in three males and one female in group 4), kidneys (hyaline casts at minor degrees of severity were noted in all treated groups of males and corticomedullary tubular basophilia at minor degrees of severity were increased in incidence in all treated groups of males. Kidney weights were also increased at 10 mg/kg in males and females at theend of the treatment period), urinary bladder (urothelial hyperplasia was recorded in three group 3 minimal or slight and all five group 4 minimal to moderate, males. In females this finding was present in all five group 4 females at minimal or slight degree), prostate gland (increased apoptosis/single cell necrosis was noted in one group 3 minimal and five group 4 males at slight degree), adrenal glands (diffuse cortical hypertrophy at minor degrees of severity was noted in some group 3 and 4 males and group 4 females), mesenteric (macrophage foci at minor degrees of severity were increased in incidence in all lymph node treated groups of both sexes) and skeletal muscle (myofiber degeneration at slight or moderate degree was noted in group 3 and 4 males and in group 4 females).

The skeletal muscle fibre degeneration in males and females at 10 mg/kg occurred in the absence of any supportive clinical signs or changes during functional observation tests or in motor activity, which indicates that the motor-function integrity of rats is not adversely affected. Also, this finding was fully reversible in males within 14 days following the last treatment, and was also seen in three male control animals. Therefore, this lesion was considered not to be adverse in nature within the context of this study.

After a fourteen day recovery period the following findings remained in evidence in group 4 males at increased incidence in
lungs (alveolar macrophage foci at minor degrees), liver (diffuse midzonal/centrilobular hypertrophy at minor degrees), kidneys (hyaline casts at minimal degree and corticomedullary tubular basophilia at minor degrees), urinary bladder (urothelial hyperplasia was recorded in three group 4 animals at minimal or moderate degree), prostate gland (increased apoptosis/single cell necrosis was noted in three group 4 males at minimal degree), and adrenal glands (diffuse cortical hypertrophy at minimal degree was seen in two group 4 rats).

After a twenty-eight day recovery period the following findings remained in evidence in group 4 males at increased incidence in
lungs (alveolar macrophage foci were noted at minimal or slight degree in three group 1 and at a moderate degree in two group 4 animals) and
urinary bladder (urothelial hyperplasia was recorded in two group 4 animals at minimal or slight degree).

Most of the above listed findings were recorded at minor degrees of severity and at incidences slightly above spontaneous background levels, and were essentially recovered within a 14- or 28-day recovery period. Therefore, these morphological lesions were considered no to be adverse in nature. However, the urothelial hyperplasia of the urinary bladder in group 3 (males) and 4 (males and females) at the end of treatment and persisting until the end of the 28-day recovery period was considered to be adverse in nature, given that it could not be excluded that this finding has preneoplastic potential. the duration of this study however is too short to substantiate this. No underlying mechanistic cause for urothelial hyperplasia of the urinary bladder could be established based on the examinations conducted in this study. The test substance has corrosive potential (information provided by the sponsor), and hence it is conceivable that this hyperplasia occurred in response to prolonged irritation and would therefore be adaptive in nature. However, this finding appeared not fully reversible within a 28-day treatment period in males (recovery in females was not examined). No evidence of a treatment-related inflammatory infiltrate in the bladder was noted (but irritation could occur without such infiltrate). Also, in case of an adaptive response this finding may be expected to have resolved within this period. It should be noted that females that underwent a longer duration did not show urothelial hyperplasia at higher severity grades than males.

The increased apoptosis/single cell necrosis noted in three males at 10 mg/kg was noted at minimal degree only, and there were no morphological abnormalities in other male primary or secondary reproductive organs. Furthermore, there was no evidence of reproductive or developmental toxicity within this study, and the finding was fully reversible within a 28-day treatment-free recovery period. Therefore, this prostate lesion in not to be regarded as an indicator of possible reproductive toxicity and in therefore considered not to be adverse in nature. According to expert judgement (Samuel M. Cohen, 2012) MTDID 25575 (trade name Clearlink 1000)is a highly irritating chemical that produces urothelial toxicity when administered by oral gavage for twenty-eight days. Clearlink 1000 is non-DNA reactive and non-genotoxic in vivo and it is likely that the non-genotoxic mode of action involves cytotoxicity and regeneration secondary to excretion and concentration of the highly irritative chemical and/or metanoite in the urine. The urothelial hyperplasia is an adaptive response to this irritation.

No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, food consumption and macroscopic examination).

Reproductive results:
No reproduction toxicity was observed up to the highest dose level tested (10 mg/kg).

Developmental results:
No developmental toxicity was observed up to the highest dose level tested (10 mg/kg).

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) was derived:
Parental NOAEL: 1 mg/kg/day (males) or 3 mg/kg/day (females), based on urothelial hyperplasia of the urinary bladder.
Reproduction NOAEL: at least 10 mg/kg/day
Developmental NOAEL: at least 10 mg/kg/day

The results of this study would not lead to the classification for repeated dose toxicity according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.