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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
As additional examinations ophthalmology, water intake measurements, urinalyses and histopathological evaluation of some organs not required in the OECD 407 Guideline (aorta, esophagus, femur, pancreas, pituitary and salivary glands) were performed.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tercarol 5903
- Molecular weight: Mn = 340 g/mole
- Physical state: liquid (red viscous)
- Content: > 99%
- Storage : room temperature under light protection

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan GmbH
- Age at delivery: about 5 weeks
- Weight at study initiation: males: 178 (160-203)g, females: 147 (138-165)g
- Housing: group housed (3 or 2 rats/cage)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 55 ± 5%
- Air changes (per hr): = 10 passages/hour
- Photoperiod (hrs dark / hrs light): 12 hours rhythm

IN-LIFE DATES: From: November 8, 2007 To: December 7, 2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: ethanol/solutol/demineralized water, 1/4/5 (w/w/v)
Details on oral exposure:
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content of the test item was assumed to be nominally 100% for calculation.
Before the start of the study formulations containing the test item in concentrations
of 1 mg/mI and 100 mg/ml were analyzed to determine stability of the test
item in the vehicle. Stability was analytically verified for a period of 7 days. No tests on homogeneity were done because the formulations were clear solutions. The test item content (all concentrations including vehicle control formulation) were checked at begin and near termination of the study.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
40, 160 and 640 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
five
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels used were based on the results of a two-week pilot study (0, 100, 300 or 1000 mg/kg) and a one week pilot study (0, 500, 750 mg/kg). During these studies Wistar rats (3 males and 3 females per dose group) received the test substance in ethanol/solutol HS 15/demineralized water (1/4/5). No c1inical findings or relevant effects on body weight development were observed in this study.

In the two-week pilot study the following test substance-related effects were seen:
At 1000 mg/kg -One of 3 males and 3 of 3 females died within 3 days.
-Body weight gain was 33% reduced in surviving males.
-Relative liver weights increased 59% in males.
-Relative spleen weights decreased 22% in males.
At 300 mg/kg -Relative liver weights increased 32% in females.
-Enlarged livers were seen in females.
-Relative liver weights increased 11% in males.
-Relative spleen weights decreased 10% in males.

There was no morphological correlate to the increase in liver weights at 300 mg/kg.
There was no dose-dependent decrease in body weight gain up to 300 mg/kg.

In the one-week pilot study the following test substance-related effects were seen:
At 750 mg/kg -One of 3 females died within 3 days.
-Body weight gain was reduced by 23% in surviving females and by 13% in males.
At 500 mg/kg -Body weight gain was reduced in females by 23%.

Positive control:
historical baseline data

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes (incl. Open Field Observations (OFO))
- Time schedule: once before start and once weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examination: at study begin and at termination
- How many animals: all dose groups incl. controls at study begin and control and high dose at termination

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once, day 28
- How many animals: all dose groups incl. controls

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once, day 28
- How many animals: all dose groups incl. controls

URINALYSIS: Yes
- Time schedule for examinations: once, day 27
- How many animals: all dose groups incl. controls

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: FOB: once, day 22-23 (absolute); MA: once, day 20-21 (absolute)
- Dose groups that were examined: all dose groups incl. controls
- Battery of functions tested: Functional Observational Battery (FOB); Motor Activity (MA)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all dose groups and controls)

ORGAN Weights:
Adrenals, brain, epididymides, heart, kidneys, Liver, Ovaries, Spleen, Testes, Thymus, uterus

Fixed organs:
Adrenals, aorta, brain (cerebrum, cerebellum, ponslmedulla), epididymides, esophagus, eyes, eyelids, extraorbital lachrymal glands, femur, harderian glands, head (with nasal and paranasal cavities), heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum and remaining intestine), kidneys, larynx, liver, lymph nodes (mandibular, bronchial/hilus, and mesenteric), lung, mamma, optical nerves, ovaries, oviducts, pancreas, pharynx, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulating glands), skeletal muscle (thigh), skin (mammary and muzzle), spinal cord (cervical, thoracic, lumbar), spleen, sternum, stomach (forestomach and glandular stomach), testes, thymus, thyroids (including parathyroid glands), tongue, trachea, ureter, urethra, urinary bladder, uterus with uterine cervix, vagina, Zymbal’s glands and all organs or tissues with macroscopic findings.

HISTOPATHOLOGY: Yes (high dose group and controls)
- Microscopic: Adrenals, aorta, brain (cerebrum, cerebellum, brain stem), epididymides, heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum and remaining intestine), kidneys, lung, lymph nodes, ovaries, oviducts, pancreas, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulating glands), skeletal muscle (thigh), skin (mammary and muzzle), spinal cord (cervical, thoracic, lumbar), spleen, stomach (forestomach and glandular stomach), testes, thymus, urinary bladder, uterus with uterine cervix, and all organs or tissues with macroscopic findings. Slides were prepared from the first five animals of all groups and evaluated from the control and the high concentration group.

HISTOPATHOLOGY: Yes (all dose groups and controls)
- Microscopic: pituitary gland, esophagus, femur, liver, sternum with bone marrow, trachea and thyroids glands
Statistics:
Statistical evaluations on body and organ weight data were done using the Dunnett test in connection with a variance analysis. Evaluating biochemical parameters an analysis of variance followed by a Dunnett test, an adjusted Welch test or a Kruskal-Wallis test was performed [see details in Section 7.7.9 (page 209), 7.7.10 (page 224), 7.7.11 (page 241) and 7.7.12 (page 258), following the tables of the respective individual values]. For all these tests SAS® routines were used.
All variables that were not dichotomous were described by sex, dose group and time point using appropriate measures of central tendency (mean, median) and general variability (standard deviation, minimum, maximum).
For the statistical evaluation of samples drawn from continuously distributed random variables three types of statistical tests were used, the choice of the test being a function of prior knowledge obtained in former studies. Provided that the variables in question were approximately normally distributed with equal variances across treatments, the Dunnett test was used, if heteroscedasticity appeared more likely, a p value adjusted Welch test was applied. If the evidence based on experience with historical data indicated that the assumptions for a parametric analysis of variance cannot be maintained, distribution-free tests in lieu of ANOVA were carried out, i.e. the Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon tests (U tests), if appropriate.
With respect to data collected in the functional observational battery categorical variables were analyzed with a repeated measures analysis of variance followed by a one-way analysis of variance using the SAS procedure PROC CATMOD. As part of the one-way analysis, contrasts were performed to compare the results of each treated group with those of the control group. The logic of the analysis plan for continuous variables was analogous to that of the categorical variables, but using the SAS procedure PROC GLM.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:

One female of the 640 mg/kg dose group was found dead on Day 3. At cage-side observations (including open field observations) at 160 mg/kg one and at 640 mg/kg two males exhibited reduced motility and abdominal position during day 5 to 8. No findings in the females of all dose groups. At detailed clinical observations done weekly no findings attributable to the test substance were seen up to and including 640 mg/kg.

BODY WEIGHT AND WEIGHT GAIN:

At 640 mg/kg males exhibited body weight depression resulting in a 11% lower mean body weight on day 28. No effect on body weight and weight gain was observed in female rats up to and including 640 mg/kg bw.

WATER CONSUMPTION:

At 640 mg/kg an increase in water intake was seen in both sexes. The intake per kg body weight was increased 16% compared to the controls.

HAEMATOLOGY:

The hemoglobin concentration as well as MCV and MCH were decreased statistically significantly at 640 mg/kg in males. In females erythrocytes, hemoglobin and hematocrit decreased dose-dependently beginning at 160 mg/kg. The thrombocytes were increased partly statistically significantly at 640 mg/kg in both sexes. The HQUICK means decreased in females beginning at 160 mg/kg. At 640 mg/kg the count of leucocytes and neutrophils increased in males and/or females. Possibly related to the liver and general toxicity seen at 160 and/or 640 mg/kg these effects are interpreted to be of toxicologically relevance.

CLINICAL CHEMISTRY

The CK activity decreased in 640 mg/kg males and the GGT activity increased in 640 mg/kg males and females. The APh activity was in the 2s range in treated males and decreased in 160 and 640 mg/kg females.
In females the cholesterol concentration was increased at 160 and 640 mg/kg and that of glucose at 640 mg/kg.

URINALYSIS:

Increased urine excretion at 160 mg/kg and 640 mg/kg in females and at 640 mg/kg in males. Beginning at 160 mg/kg urine samples were brown-reddish discolored. Since pathomorphological correlates were lacking, the increase in urine excretion was not indicative for a toxic effect.

NEUROBEHAVIOUR:

At FOB measurements males and females receiving 640 mg/kg showed salivation. 640 mg/kg males (3/5) exhibited additionally reduced movement, which was interpreted to be based on reduced general condition rather than neurotoxicity.

ORGAN WEIGHTS:

Absolute and relative liver weights increased dose-dependently and partly statistically significantly beginning at 40 mg/kg in both sexes (only marginal at 40 mg/kg). In ascending doses the relative weights increased by 3%, 17% and 43% in males and 8%, 34% and 91% in females.
Furthermore, absolute (17%) and relative (15%) weights of the uterus were found to be increased at 640 mg/kg.

GROSS PATHOLOGY:

At 160 mg/kg and above both sexes revealed liver changes (swelling, enlargement, distinct lobulation, discoloration). Enlargement of the thyroid glands was observed in single males at 160 mg/kg and above, and in females at 640 mg/kg.

HISTOPATHOLOGY:

At 160 mg/kg and above males and females showed up to moderate periportal hepatocellular change. Periportal liver areas were increasingly characterized by hypertrophy, discoloration, nuclear pleomorphism and/or an increased number of liver cells with disorganisation. Females demonstrated a higher tendency to binucleated hepatocytes. Slight activation and/or proliferation of bile ducts were detected in both genders at 640 mg/kg. Portal inflammation was elevated in females beginning at 160 mg/kg. Kupffer cell foci were reduced in males at 640 mg/kg. Periportal fat deposition increased in males and more pronounced in females at 40 mg/kg and above, but without adverse impact.
In the thyroids, follicular cell hypertrophy/hyperplasia and colloid decrease up to a moderate degree were diagnosed in both genders at 160 mg/kg and above. In the pituitary gland, pale hypertrophic, so-called thyroidectomy cells increased in males at 160 mg/kg and above.

Effect levels

Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Histopathological changes in the liver and thyroid.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

In a repeated dose oral toxicity study in rats (Wistar, OECD TG 407), o-Toluene diamine, propoxylated was adimistered via gavage to 5 rats/sex/dose at 0, 40, 160, 640 mg/kg bw for 4 weeks. One female of the 640 mg/kg group was found dead on day 3. The behavior and clinical appearance of the rats were not influenced by the treatment at 40 mg/kg in males and up to and including 640 mg/kg in females. Beginning at 160 mg/kg few males exhibited signs of reduced health conditions such as reduced motility and/or abdominal position. The body weight gain was not affected up to and including 160 mg/kg in males and up to 640 mg/kg in females. At 640 mg/kg up to 11% body weight depression was seen in males. The food intake was not changed up to and including 640 mg/kg. The water intake was slightly increased at 640 mg/kg in both sexes. Functional Observational Battery (FOB) measurements males and females receiving 640 mg/kg showed salivation. 640 mg/kg males exhibited additionally reduced movement, which was interpreted to be based on reduced general condition rather than neurotoxicity. Motor and Locomotor Activity (MA/LMA) tests did not indicate neurotoxicity up to and including 640 mg/kg. Blood parameters were altered in 640 mg/kg males and in females beginning at 160 mg/kg. No remarkable changes occurred in enzyme activities up to and including 160 mg/kg in males and at 40 mg/kg in females. Morphological indications of liver toxicity at 160 mg/kg and above in both sexes. In the thyroids, follicular cell hypertrophy/hyperplasia and colloid decrease up to a moderate degree were diagnosed in both sexes at 160mg/kg. This change is regarded to be secondary to the liver changes and is frequently observed in rats following induction of microsomal enzymes. This mechanism is regarded to be probably of no relevance to humans. In the pituitary gland, pale hypertrophic, so-called thyroidectomy cells increased in males at 160 mg/kg and above. Based on these results the NOAEL was considered to be 40 mg/kg bw and day for both sexes.