Registration Dossier

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study, however spacing of doses is not adequate and historical control data is missing
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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): TP-90B Rubber Chemical
- Analytical purity: 97% (v/v)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: not described
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: Males 361.23-362.49 g, females 262.20-262.98

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
Test substance was suspended in an aqueous solution of 0.5% CMC at concentrations of 1.0, 10.0, 80.0 mg/ml. Preparations were prepared daily
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
at least ca. 6 weeks:
For males, treatment commenced when the males were approximately 12 weeks old and continued for two weeks prior to pairing, during pairing until mating was confirmed, and up to and including the day before sacrifice.
For females, treatment commenced when they were approximately 12 weeks old and continued for two weeks prior to pairing through the mating and gestation periods, up to day 3 of lactation. Dams and offspring were sacrificed on Day 4 post-partum.
Frequency of treatment:
once per day, 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 100, 800 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CLINICAL SIGNS
Once before the commencement of treatment, and at least once per week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behavior and effects on the autonomic nervous system (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern).
During the gestation periods, the observations were carried out on Days 1, 7, 14, and 20.

MORTALITY
Twice per day

BODY WEIGHT
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to pairing, and on gestation days 0, 7, 14, and 20. Dams were also weighed on days 1 and 4 post-partum, with the exception of one mid-dose female, which was weighed on days 2 and 5 post-partum.

FOOD CONSUMPTION
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation days 0, 7, 14 and 20, and on days 1 and 4 post-partum, with the exception of one mid-dose female where food consumption was measured on days 2 and 5 post-partum.

HEMATOLOGY
Measurements performed on blood samples were as follows: hematocrit; hemoglobin; red blood cell count; mean RBC volume; mean corpuscular hemoglobin; mean corpuscular hemoglobin concentration; white blood cell count; differential leukocyte count (neutrophils, lymphocytes, eosinopils, basophils, monocytes, large unstained cells): abnormalities of the blood film; platelets; prothrombin time; partial thromboplastine time

BIOCHEMISTRY
Clinical chemistry parameters investigated included: alkaline phosphatase; aminotransferase; aspartate aminotransferase; urea; creatinine; gamma glutamyl transferase; globulin and A/G ratio: fasting glucose; total bilirubin; total cholesterol; total protein; albumin; sodium; phosporus; potassium; calcium; chloride; total glycerides.

Sacrifice and pathology:
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):

- Macroscopic: The following organs from all adult animals in all groups were dissected free of fat and weighed: adrenals, brain, epididymides, heart, kidneys, liver, spleen, thymus, testes.

- Microscopic: The tissues listed, as follows, were examined in all adult animals: corpora lutea, abnormalities (gross lesions), adrenals (paired), bone marrow (from the sternum), brain, caecum, coagulating glands (paired), colon, duodenum, epididymides (paired), esophagus, heart, ileum, jejunum, kidneys (paired), liver, lymph nodes (cervical), lymph nodes (mesenteric), lungs, ovaries with oviduct (paired), prostate, pituitary, rectum, sciatic nerve, seminal vescicles, spinal column with spinal cord, spleen, stomach, thymus, thyroid, trachea, testes (paired), urinary bladder, uterus with cervix, vagina.
Other examinations:
- Pre- and post-dose observations:
Examination of individual animals for signs of reaction to treatment was carried out daily. Starting on day 1 until day 14 of dosing, all animals were observed prior to dosing, immediately after dosing, and at 1, 2 and 3 hours after dosing. From day 15 until study termination, observations were made prior to dosing, immediately after dosing, and 15 minutes and 1 hour after dosing.

- Sensory reactivity and grip strength:
Once during the study, in week 5 of treatment, 5 males were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g., auditory, visual and propioceptive stimuli), and an assessment of grip strength was also performed. Once during the study, on day 20 post-coitum, 5 females were selected for the same tests.

- Motor activity:
Once during the study, on week 4 of treatment, 5 males were randomly selected from each group and the motor activity was measured by an automated activity recording device. Measurements were performed using a computer-generate random order. On day 20 post-coitum, 5 females were selected for the same activity.
Statistics:
For continuous variables, the significance of the difference amongst group means was assessed by analysis of variance. Differences between the treated group and the control group were assessed by Dunnett's test using a pooled error of variance. The homogeneity of the data was assessed by Bartlett's Test before Dunnett's Test was performed. If the data were found to be inhomogeneous, a modified t-test (Cochran and Cox) was applied. Statistical analysis of histopathological findings was not carried out by means of the non-parametric Kolmogorov-Smirnov test.
The non-parametric Kruskal-Wallis analysis of variance was used for all other parameters. Intergroup differences between the control and the treated group were assessed by the non-parametric version of the Williams' test.

Results and discussion

Effect levels

Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

MORTALITY AND TIME TO DEATH

One mid-dose female was sacrificed, on day 0 post-partum, for humane reasons, since signs of difficult delivery were noted. No other animals died prior to study termination.

CLINICAL SIGNS

Weekly physical examinations including detailed clinical signs with neurotoxicity assessment did not show any signs which could be correlated to the treatment with the test item in males or females in any group during the treatment period.

In the high dose group ataxia, semi-closed eyes and hunched posture were the principal signs were observed at 2 hours post-dose in some high dose males on day 7 of the study. Thereafter, the above signs were noted occasionally until day 10 of the study. Starting from day 30 until day 35 of the study, ataxia, reduced activity and salivation were also noted within 15 minutes of dosing in the same male group.

In high-dose females pronation, ataxia, semi-closed eyes, reduced activity, hunched posture and lethargy was observed 2 and 3 hours after treatment. In addition, twitches, piloerection and salivation were also noted 15 minutes (starting from day 15 of the study) and 1 hour after dosing in a few high dose females throughout the study.

BODY WEIGHT GAIN

Body weight and body weight gain were unaffected by treatment in the males throughout the study and in females before pairing. Slight decreases, not statistically significant, in body weight and body weight gain were observed in the high dose females during the gestation period when compared to controls. No differences were noted in females of any treatment group during the post-partum period when compared to controls.

FOOD CONSUMPTION

Food consumption remained comparable between control and treated groups in both sexes before pairing. A statistically significant reduction in food consumption was noted in high dose females on day 7 post-coitum. A similar reduction (not statistically significant) was noted on day 14 post-coitum in the same group.

CLINICAL CHEMISTRY

No toxicologically significant change in any clinical chemistry parameter occurred at any dose level.

A statistically significant decrease in alkaline phosphatase level was observed in the high dose male group. Since this change was minimal, was decreased compared to the control group and occurred in only one sex, it was not considered toxicologically significant. A statistically significant decrease in alanine aminotransferase level was noted in the low dose male group when compared to controls. This decrease was considered incidental to treatment since no dose response was evident.

Statistically significant decreases in urea were seen in the low and high dose males compared to controls. These changes were not considered treatment-related since the changes were decreased relative to controls and there was no evident dose response. Statistically significant changes in creatinine and inorganic phosphorus levels were observed in the high dose male group. These changes were not considered treatment-related since the values were decreased compared to controls, the magnitude of the change was small and they occurred only in one sex.

Urea level was also statistically significantly decreased in the low dose male group. This change was not considered treatment-related since the effect was decreased, there was no obvious graded dose response at the mid- and high dose groups and the effect occurred in only one sex.

No differences in clinical chemistry parameters were noted between control and treated females.

HEMATOLOGY

No toxicologically significant changes in hematology occurred in this study. Statistically significant decreases in prothrombin time were observed in the high dose male group. This change was considered incidental and not treatment-related since it was seen in only one sex and the magnitude of the effect was low.

Statistically significant decreases in neutrophil percentage were observed in the high dose females. This change was considered incidental and not treatment-related since it was seen in one sex only and no other changes were seen in the white blood cell differential counts.

MOTOR ACTIVITY AND SENSORY REACTION TO STIMULI

No treatment-related effects on motor activity and sensory reaction to stimuli were seen in either sex at any dose.

An increased incidence, with statistical significance, in motor activity measurements of males was noted in the low and mid-dose groups. No increase was observed in the high dose group and there was no increased magnitude of the response of the mid-dose compared to the low dose rats. The increased activity in the low and mid-dose groups was considered incidental to treatment with the test item. No differences were noted in treated females when compared to controls.

ORGAN WEIGHTS

Treatment-related organ weight changes occurred in the liver of high dose males and females.

Statistically significant increases in absolute and relative liver weights were observed in the high dose males when compared to controls. Increases in absolute (not statistically significant) and relative (statistically significant) liver weights were also noted in only the high dose females.

Statistically significant increases in absolute and relative kidney weights were noted in the mid-dose male group only. Since the absolute and relative kidney weights of the high dose males were not affected (i.e., no apparent dose response), there was no corresponding increase in females, and no corresponding microscopic change in this tissue, the kidney weight changes were considered not related to treatment with the test item. Slight, but statistically significant increases in absolute and relative adrenal weights were observed in the high dose female group. This change was considered incidental and not treatment-related since there was no corresponding microscopic histopathologic change in this tissue and similar effects were not seen in the high dose male group.

GROSS PATHOLOGY

No macroscopic change was reported at necropsy in the examined organs/tissues of the animals killed at termination that could be considered related to the administration of the test substance. The recorded changes were considered to be incidental or spontaneous in origin.

HISTOPATHOLOGY

No microscopic changes clearly related to test substance administration were observed in male or female rats given 10 or 100 mg/kg bw/day of the test article.

Diffuse hepatocellular hypertrophy (enlarged hepatocytes in all areas of the lobules) occurred in male and female rats of the high dosage group and was considered to be treatment-related.

Focal germ-cell depletion and degeneration occurred in one testis (unilateral) of each of four different male rats of the high dosage group. Exfoliated spermatogenic cells were observed in the epididymis in three of these high dose rats and in one additional high dose rat without testicular changes. The significance of this finding was uncertain. Systemic toxicity usually did not affect only one of a paired organ.

All other microscopic changes were considered to have occurred spontaneously and to be unrelated to test substance administration. These changes generally occurred at single, low, or similar frequencies among the groups and their type, incidence or severity was not influenced by compound administration.

Applicant's summary and conclusion