Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: OECD Guideline 408 compliant study with spermiological examination
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 December 2013 - 05 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
03 October - 06 November 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
test item was administered for 14 days only
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BIOAGRI Laboratórios – DF/ Brazil
- Age at study initiation: 7 weeks
- Weight at study initiation: Mean body weight was 381.2 ± 13.1 g for males and 233.8 ± 9.0 g for females
- Housing: Animals were housed individually in polypropylene rodent cages (41x34x19cm) with wire mesh tops
- Diet: Animals were fed Supralab diet for rodents (supplied by Alisul Alimentos S.A, Rio Claro, Brazil), ad libitum
- Water: Filtered drinking water was supplied by CAESB (Companhia de Saneamento Ambiental do Distrito Federal), ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.2-22.0 ° C
- Humidity: 30.2–70.0 %
- Air changes: 10 to 20 air changes per hour
- Photoperiod: 12 h dark / 12 h light
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- For each dose level, the appropriate amount of the test item was weighed into a pre-calibrated beaker, added to the ground plain ration and turned into flour. The test diets were prepared weekly and checked for concentration and homogeneity.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability analysis: Stability analysis of the test item in feed was performed prior to the initiation of the study. The test item in the diet is stable for 8 days.
Concentrations and Homogeneity: The concentration and homogeneity of the test item for each group was determined for each new preparation.
Acceptance criteria at each time-point: Actual concentration: nominal value ±10 %. The acceptance criteria were met.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
800, 3000 and 12000 ppm in the diet (corresponding to 67, 213 and 635 mg/kg bw/day in males and 71, 234 and 802 mg/kg bw/day in females)
Basis:
nominal in diet
No. of animals per sex per dose:
5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: In a non-GLP palatability study performed on 5 animals/sex/group, animals were fed with preparation at 20000 ppm test item for 5 days. Food consumption in males almost returned to control level on the fourth day of treatment but females still ate almost 50 % less than controls. Based on these results, the following three dose levels have been selected: 800, 3000 and 12000 ppm.
- Rationale for animal assignment: Randomization was performed one day prior to the beginning of the treatment period. Prior to randomization, the animals were weighed and a detailed clinical examination was performed. The required number of animals was selected according to their health status and recorded body weight (± 10 % of the mean body weight of each sex). Selected animals were randomly assigned to the test groups using a computerized randomization procedure.
Positive control:
Not applicable
Observations and examinations performed and frequency:
DAILY OBSERVATIONS: Yes
- Time schedule: During acclimation period each rat was observed daily in the cage for signs of ill health, morbidity and/or mortality. During treatment period, all animals were observed for morbidity and/or mortality at least twice daily (in the morning and in the afternoon on working days), once daily on weekend. All animals were also observed once daily for clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were submitted to a careful physical examination by a veterinarian, outside the home cage, at approximately the same time of day, prior to the initiation of treatment and once weekly during the treatment period paying particular attention to:
- changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size and unusual respiratory pattern);
- changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backwards).

BODY WEIGHT: Yes
- Time schedule for examinations: Each rat was weighed at the end of the acclimation period and prior randomization, on the first day of treatment and then daily during the treatment period. The rats were weighed on the day before the scheduled necropsy (not fasted) and also on the day of necropsy (fasted).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was evaluated daily at the same time body weights were recorded for all animals during the treatment period. Food consumption was calculated per animal and per day.
- Mean test substance intake for each sex-group was calculated daily, individually, according to the food consumption and expressed as ppm.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood collection was done by cardiac puncture at the end of the study.
- Samples were collected for complete blood count (EDTA sample tubes), blood coagulation (samples in tubes with sodium citrated) and for clinical biochemistry (samples in tubes without anticoagulant).
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes; overnight fasting
- How many animals: 5/sex/dose
- Parameters checked for haematology: Red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, total white blood cell count, differential white blood cell counts (lymphocytes, monocytes, eosinophils, basophils, band neutrophils and segmented neutrophils), clotting parameters (prothrombin time and activated partial thromboplastin time)
- Parameters checked for clinical chemistry: Glucose, blood urea nitrogen, creatinine, total protein, globulin, albumin, albumin/globulin ratio, total bilirubin, calcium, total cholesterol, sodium, potassium, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and triglycerides

URINALYSIS: Yes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; A complete gross examination was performed on all animals, after euthanasia by carbon dioxide inhalation and bleeding. The necropsy consisted of a systematic gross examination of general physical conditions, orifices, external and internal organs and tissues. The following organs from each animal were weighed during necropsy:
Adrenals (right and left), brain, heart, kidneys (right and left), liver, pituitary gland, spleen, thymus, thyroids (with parathyroids), testis (right and left), epididymides (right and left), prostate, seminal vesicles, ovaries (right and left) and uterus

HISTOPATHOLOGY: No
Other examinations:
None
Statistics:
Quantitative variables such as mean body weight and mean body weight change, food consumption, urinalysis, hematological assessment (hemogram, leukogram, clotting time, and biochemistry) were analyzed by One-Way Analysis of Variance (ANOVA), followed by Dunnett’s or Wilcoxon tests if significance was detected. For qualitative or non-parametric data such as clinical signs, macroscopic and microscopic findings, comparison was carried out using the Chi-Square Test. The level of significance was set at 5 % and the statistical program used was SAS Software (SAS Institute Inc., Cary, NC).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- No mortality or clinical signs were observed during the study.

BODY WEIGHT AND WEIGHT GAIN
- Test item produced dose-related effects on body weight (BW) and body weight gain (BWG). BW in males differed statistically from control groups at mid and high dose; accordingly, the overall mean BW values were statistically affected (males -9.6 and -21.3 % and females -9.1 and -13.7 % at 3000 and 12000 ppm, respectively). Mean BWG in males and females were sometimes statistically significantly lower than control group at mid and high doses leading to an overall mean BWG statistically significantly lower at 3000 and 12000 ppm. At low dose both males and females mean BW and BWG were reduced too but not statistically significantly; although moderate in degree, the decrease in BWG (-18.2 and -29.2 % in males and females, respectively) cannot be obviate.

FOOD CONSUMPTION (if feeding study)
- In all treated groups, the test item produced a dose-related decrease in food consumption, mostly at mid and high doses in males and females, affecting statistically the overall food consumption in these groups (males: -24.2 and -49.5 %; females: -21.3 and -36.5 % at mid and high doses, respectively). These findings correlate with the lower BW and BWG in males and females at these dose levels. Small differences in food consumption were observed at low dose in males and females (-3.5 and -5.2 %, respectively). While a relation to treatment of the lower food consumption at 800 ppm cannot be excluded, this change was minimal, within the range of common variation of this parameter, and is therefore considered to be of no toxicological relevance.

HAEMATOLOGY
- Treatment-related finding was noted in hematocrit in male and female treated rats. Decreased mean values were dose proportional, more pronounced in females than in males and statistically significant at high dose (-6.7 and -11.8 % in males and females, respectively). Nevertheless, all mean and individual values are within the control range for this species of rat and age. Other differences between controls and treated groups were minimal, either not statistically significant or not dose related and within normal control range.
- Changes in the leukogram were observed at 12000 ppm. Statistically significant decrease in segmented neutrophils was observed in males and females of the high dose group (-63.0 and -48.9 %, respectively) when compared to the control groups; this was associated to moderate lower values in most leukocytes.
- Statistically significant lower mean Prothrombin Time (PT) and higher mean Activated Partial Thromboplastin Time values were noted in treated females when compared to the control. Mean PT values were small, dose proportional and statistically significant at high dose (-5.8, -6.7 and -9.2 % at 800, 3000 and 12000 ppm, respectively) while APTT were moderate in magnitude, not dose proportional and statistically significant at mid and high doses (+0.7, +36.6 and +18.6 % at 800, 3000 and 12000 ppm, respectively). Lower PT and higher APTT results were due to slightly higher values in PT and lower APTT in the control group in accordance with standard ranges (PT: 14-20 and APTT: 12-18), then without abnormal biological considerations.


CLINICAL CHEMISTRY
- Blood urea nitrogen and total protein were the only parameters that increased with dose and reached statistical significance at the high dose, but only in females. Slightly higher values were observed in males of the high dose group but not statistically significant. Total bilirubin was statistically higher than controls at high dose for both males and females, but this increase was not dose related at lower dose levels. Total cholesterol was statistically higher in males of the high dose only, without dose relationship as a statistically significant lower value was observed at 3000 ppm. Thus, the only potential toxicological significant effect in clinical chemistry parameters was the increase in total bilirubin as it was dose-related, reached statistical significance at high dose and was observed in both sexes.


ORGAN WEIGHTS
- Statistically significant absolute organ weight decreases were noted for heart, spleen and thymus for both male and female rats, seminal vesicle for males, and adrenal and ovaries for females at 12000 ppm. Only the decrease in seminal vesicle weight was confirmed in organ weight relative to bodyweight, although treated males had statistically significant lower mean bodyweights. This decrease was associated to statistically significant decrease in testis and epididymides bodyweight-relative weights at the high dose. The other toxicological significant effect observed in males and females was the dose-related and statistically significant increase in absolute and bodyweight-relative liver weight at mid and/or high dose levels.

GROSS PATHOLOGY
- No macroscopic abnormalities were observed in treated animals.
Dose descriptor:
NOAEL
Effect level:
800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicity effects were observed at 800 ppm.
Critical effects observed:
not specified

None

Conclusions:
Under these test conditions, the No Observed Adverse Effect Level (NOAEL) of Tetrapropylene benzene was considered to be 800 ppm in rats. Based on these results, the highest dose of the test item recommended as 3000 ppm for the 90-day repeated dose toxicity study.
Executive summary:

In a repeated dose oral toxicity study conducted similarly to the OECD Guideline 407 and in compliance with GLP, test item, Tetrapropylene benzene was administered daily by feed to groups of Sprague-Dawley rats(5/sex/dose) at dose-levels of 800, 3000 and 12000 ppm in the diet (corresponding to 67, 213 and 635 mg/kg bw/day in males and 71, 234 and 802 mg/kg bw/day in females) for 14 consecutive days in order to select dose levels for 13-week repeated dose toxicity study. The control group received the feed alone. Examinations during the study included: mortality, clinical signs, body weight, food consumption, haematology, blood chemistry, organ weights and macroscopic examination.

 

No mortality or clinical signs were observed during the study. Body weight and body weight gain were statistically significantly reduced in a dose depend manner, in rats treated at 3000 and 12000 ppm when compared to control. These findings correlate with the decrease in food consumption, therefore they are probably due to the low palatability of the test substance. Reduction in hematocrit at 12000 ppm was observed in males and females. Statistically significant decrease in segmented neutrophils was observed in males and females of the high dose group, associated with moderate lower values in most leukocytes counts. The only potential toxicological significant effect in clinical chemistry parameters was the increase in total bilirubin as it was dose-related, reached statistical significance at high dose and was observed in both sexes. Statistically significant decreases were observed in mean absolute weights of several organs in males and/or females at high dose but only seminal vesicle decrease was confirmed in organ weight relative to bodyweight. This decrease was associated to statistically significant decrease in testis and epididymides weights relative to bodyweight at the high dose. The other toxicological significant effect observed in males and females was the dose-related and statistically significant increase in absolute and bodyweight-relative liver weight at mid and/or high dose levels. All these alterations, especially decreases in body weight and body weight gain, suggest treatment-related systemic toxicity at mild and high doses.

 

Under these test conditions, the No Observed Adverse Effect Level (NOAEL) of Tetrapropylene benzene was considered to be 800 ppm in rats. Based on these results, the highest dose of the test item recommended as 3000 ppm for the 90-day repeated dose toxicity study.

Data source

Reference
Reference Type:
other: draft audited report
Title:
Unnamed
Year:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD guideline 408
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Tetrapropylene benzene (BAB)
- Physical state: Clear colorless liquid
- Analytical purity: 100 % UVCB
- Lot/batch No.: 981204
- Date of receipt: 09 April 2013
- Expiration date of the lot/batch: 23 December 2014
- Stability under test conditions: Stable at room temperature
- Storage condition of test material: Stored at room temperature; protected from light and humidity

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BIOAGRI Laboratórios – DF/ Brazil
- Age at study initiation: 7-8 weeks
- Housing: Animals were housed individually in polypropylene rodent cages (41 x 34 x 19 cm) with wire lids on top
- Diet: Animals were provided a conventional laboratory diet for Rodents (Nuvilab CR -1, Nuvital Nutrientes Ltda, Curitiba – PR, Brazil), ad libitum
- Water: Drinking water supplied by CAESB (Companhia de Saneamento Ambiental do Distrito Federal), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.3 – 23.1 ° C
- Humidity: 44.9-67.6 %
- Air changes: 10-20 air changes per hour
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- For each dose level, the test item was weighted in a beaker, added to the ground plain ration and turned into flour. The test diets were prepared each 15 days and checked for concentration and homogeneity.
Details on mating procedure:
Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability analysis: Stability analysis of the test item in feed was performed in a separate study (Study No.: 11995.020.035.13). The test item in the diet was stable for 16 days. Then, the formulation was prepared each 15 days.
Concentrations and homogeneity: The concentration and homogeneity of the test item for each group was determined for each new preparation.
Analyses of the preparations showed that they all fulfilled the acceptance criteria (i.e. nominal ± 10 %).
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Details on study schedule:
None
Doses / concentrations
Remarks:
Doses / Concentrations:
300, 1000 and 3000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
Principal group: 10 animals/sex/dose for control, pair-fed, low (300 ppm), mid (1000 ppm) and high dose (3000 ppm)
Satellite group: 5 animals/sex/dose for control, pair-fed and high dose (3000 ppm)
Control animals:
yes, plain diet
other: pair-fed control group
Details on study design:
- Dose selection rationale: Based on the results of a preliminary 14-day oral toxicity study (Study No.: 11995.306.002.13), the following three dose levels were selected: 300, 1000 and 3000 ppm; the control groups received feed alone. In this preliminary study, major toxicological effects were a significant decrease in bodyweight and bodyweight gain at 3000 and 12000 ppm, associated with a decrease in food consumption, probably due to low palatability of the preparations.
- Rationale for animal assignment: At the end of acclimation period, healthy animals were randomly assigned to the experimental groups using a computerized randomization procedure and housed individually. Prior to randomization, the animals were weighed and a detailed clinical examination was performed. The required number of animals was selected according to their health status and body weight. At the beginning of the treatment, the body weight variation did not exceed ± 20 % of the mean weight of each sex.
- Rationale for selecting satellite groups: After 90 days of treatment, the test item administration was suppressed from the satellite animals and the animals necropsied after 28 days to verify possible reversibility of test item related effects.
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
None

Examinations

Parental animals: Observations and examinations:


CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During acclimation period each rat was observed daily in the cage for signs of ill health, morbidity and/or mortality. During treatment period, all animals were observed for morbidity and/or mortality at least twice daily (in the morning and in the afternoon on working days), once daily on weekend and public holidays. All animals were also observed once daily for clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were subjected to a careful physical examination outside the home cage, at approximately the same time, prior to the initiation of the treatment and once weekly during treatment period. Signs to be recorded included:
- changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern);
- changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilation, walking backwards).

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed prior to randomization, on the day of the first administration and twice a week during the treatment period, on the day before the scheduled necropsy (not-fasted) and on the day of the scheduled necropsy (fasted).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was evaluated daily for all animals during the treatment period.
- Mean test substance intake for each sex-group was calculated daily, individually, according to the food consumption and expressed as ppm.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination was performed prior to the initiation of dosing and at the end of treatment period.
- Dose groups that were examined: Animals from all groups

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before necropsy
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes; overnight period of at least 12 h
- How many animals: Animals from all groups
- Blood samples were collected by intracardiac puncture for erythrogram (EDTA tubes), blood coagulation (sodium citrate tubes) and clinical biochemistry (tubes without anticoagulant).
- Parameters checked for haematology: Red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, total white blood cell count, differential white blood cell counts (lymphocytes, monocytes, eosinophils, basophils, band neutrophils and segmented neutrophils), clotting parameters (prothrombin time and activated partial thromboplastin time)
- Parameters checked for clinical chemistry: Glucose, blood urea nitrogen, creatinine, total protein, globulin, albumin, albumin/globulin ratio, total bilirubin, calcium, total cholesterol, sodium, potassium, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: Before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes; overnight period of at least 12 h
- Parameters checked: Volume, density, pH, smell, appearance (clarity), color, nitrites, protein, glucose, ketones, urobilinogen, bilirubin, occult blood, leukocytes, erythrocytes, epithelial cells, bacteria, mucus and crystal

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional Observational Battery (FOB) was performed on all animals during the 11th week of dosing.
- Dose groups that were examined: Animals from all groups
- Battery of functions tested:
Autonomic functions: lacrimation, salivation, palpebral closure, prominence of the eye, piloerection, respiration, urination and defecation;
Reactivity and sensitivity: sensor motor responses to approach tactile and tail flick;
Excitability: reactions to handling and behavior in an open field;
Gait and sensor motor coordination: degree of morbidity, gait pattern in an open field, aerial righting reflex and landing foot splay; and
Abnormal clinical signs: including convulsions, tremors, unusual behavior and deposits around the eyes, nose or mouth.
Sperm parameters (parental animals):
Sperm motility, morphology, enumeration of homogenization-resistant spermatids and determination of the sperm reserve
Litter observations:
not applicable
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes; A complete gross examination was performed on all animals after euthanasia by carbon dioxide inhalation. The following organs from each animal were weighed during necropsy:
Adrenals (right and left), brain, heart, kidneys (right and left), liver, pituitary gland, spleen, thymus, thyroids (with parathyroid), testis (right and left), epididymides (right and left), prostate, seminal vesicles, ovaries (right and left) and uterus

HISTOPATHOLOGY: Yes (see table)
Postmortem examinations (offspring):
not applicable
Statistics:
Quantitative variables such as mean body weight and mean body weight change, food consumption, FOB, hematological assessment (erythrogram, leukogram, clotting time) and biochemistry were analyzed by One-Way Analysis of Variance (ANOVA), followed by Dunnett’s test if significance was detected. For qualitative or non-parametric data such as urinalysis, macroscopic and microscopic findings comparison was carried out using the Chi-Square Test. The level of significance was set at 5% and the statistical program used was SAS Software (SAS Institute Inc., Cary, NC).
Reproductive indices:
Not applicable
Offspring viability indices:
Not applicable

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
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
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
not examined

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
- No clinical signs or mortality occurred during the study.

BODY WEIGHT AND WEIGHT GAIN
- No significant differences in mean body weight (BW) were observed at 300 and 1000 ppm. A statistically significant decrease was observed in mean BW and mean body weight gain (BWG) in males and females treated at 3000 ppm and in males pair-fed group when compared to the controls. In the principal groups, decreases in BW were observed in males and females treated groups and pair-fed groups, from the beginning of the treatment.
- Lower overall BWG were observed in all treated groups in a dose proportional manner: -14.4, -16.1 and -46.6 % (males) and -9, -19 and -31 % (females) of the control group at low, mid and high doses, respectively. The pair-fed group had BWG comparable to the high dose group, i.e. -50 % in males and -33 % in females of the control groups.
- In satellite males, high dose and pair-fed groups usually displayed decreases in BW and BWG throughout the treatment period, similarly to main groups. At day 91, the BW difference between both groups was -20.4 and -12.2 % when compared to the control, statistically significant only for the 3000 ppm group (and slightly lower than the corresponding principal groups). The BWG was -28 and -44 % of the control group in pair-fed and 3000 ppm groups, respectively. During the recovery period, BWG in pair-fed and 3000 ppm groups almost doubled compared to control group. The overall BW for high dose and pair-fed group at day 120 was -14.2 % (statistically significant) and -5.2 % (not statistically significant) of control group, respectively. These changes in BW and BWG in males at high dose were consistently related to their lower food consumption than control group and was confirmed with the better performance of satellites animals after day 91 when the test item was removed from the feed (recovery period).
- In satellite females groups, no statistically significant differences in BW were found among the groups although overall BW were lower in pair-fed and 3000 ppm groups than control group, with isolated statistical differences between pair-fed and control group. BWG were -29 and -21 % lower than control group during the treatment period in pair-fed and 3000 ppm groups, respectively. During recovery, BWG tripled in pair-fed group and doubled in 3000 ppm group compared to control group. Changes in BW and BWG were related to lower food consumption in animals from the principal group at high dose and in pair-fed group; more marked in males and associated to low palatability of the ration at 3000 ppm. The increase in BW and BWG in satellites animals at 3000 ppm during recovery period supported the reversibility of these alterations, although satellite males of the 3000 ppm had still statistically significant lower bodyweights than control group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Compound-related effect was observed in Food Consumption (FC) in treated animals at 3000 ppm, more pronounced in males than in females, affecting the Body Weight (BW) and Body Weight Gain (BWG). Consistent lower daily FC values were noted in high dose and pair-fed groups i.e., overall FC: -25.5 and -24.9 % in males (statistically significant) and -10.8 and -11.7 % in females (sporadically statistically significant) of control groups, respectively. No significant changes in FC were noticed at 300 and 1000 ppm.
- In satellite males, the daily FC was usually lower between control group, high dose and pair-fed group, during the treatment period and sporadically statistically significant. In satellite females, overall lower FC: -14 and -12 % were observed in pair-fed and 3000 ppm treated groups, respectively. The overall FC during the recovery period was similar between all groups. These changes were associated to the low palatability of the diet containing the test item at 3000 ppm and responsible for the BW and BWG alterations in these groups.
- The animals were exposed to 300, 1000 and 3000 ppm, corresponding to a test item intake of 24, 75 and 223 mg/kg bw/day respectively for males and 27, 91 and 264 mg/kg bw/day respectively for females. Treated satellite groups were exposed to 3000 ppm, corresponding to 249 and 271 mg/kg bw/day for males and females, respectively.

OPHTHALMOSCOPIC EXAMINATION
- No ophthalmological findings were observed.

HAEMATOLOGY
- Only statistically significant lower RBC values were seen at doses of 1000 ppm and 3000 ppm (-5 and -9.2 %, respectively) in males when compared to control. In satellite males, the decrease in RBC was not statistically significant, which were considered reversible. In females, only numerical variations were observed when compared to the control group. No statistically significant or dose-related findings were observed for all parameters.
- Lower WBC was observed in treated and pair-fed animals compared to the controls. In males it was dose related trend and statistically significant at high dose (-40.6 %). As lower, statistically significant value was found in pair-fed group (-39.6%), this finding could not be related to a test item effect. In satellite males, WBC in pair-fed animals was similar to control group, while it was still lower than control (-19 %) but not statistically significant at high dose. In female treated rats, the decrease in WBC was lower, not dose proportional and not statistically significant when compared to control. In satellite treated animals the decrease was -22.7 % of the control group but it was not statistically significant.
- No biologically significant test item-related changes were observed in clotting parameters. Small numerical variations, not dose proportional, were observed in treated and pair-fed male and female animals compared to the controls. Exceptionally higher statistically significant APTT was found only at low dose females group, therefore not test item associated.

CLINICAL CHEMISTRY
- Aspartate aminotransferase (AST) and triglycerides were decreased dose proportionally with statistical significance at 3000 ppm in males. Other changes in male’s clinical chemistry values were usually small, not dose proportional even if statistically significant. No differences were found in satellite males when compared to control, except for AST.
- Like males, only sporadic statistically significant, almost never dose-related findings were observed in females. Only total bilirubin was statistically significantly lower in all treated groups, but also in pair-fed group, showing that it was not due to the test item. Also, control value in the satellite group was comparable to principal treated groups values, confirming that these lower values were not due to the test item but to high values in the principal control group.
- No clear significant toxicological effect attributable to the test item could be identified from these analyses.

URINALYSIS
- No biological differences were observed between control and treated rats, as well as the control-fed groups. Isolated statistically significant differences occurred, all within normal reference values and in pair-fed group, showing no test item-related effect.

NEUROBEHAVIOUR
- No biologically significant test item-related changes were observed during neurotoxicological testing in male or female treated rats and pair-fed animals when compared to the control groups. In males only numerical variations were seen in open field evaluation and functional observation battery. Isolated and not test item-related statistical differences were noted in females at mid dose (principal group) and in male and female satellite control groups.

ORGAN WEIGHTS
- In males, potential compound related effects were observed at 3000 ppm in spleen, thymus, epididymides, seminal vesicles and prostate absolute weights, with statistically significant decreases recorded. No relevant differences were found in males from low and mid doses when compared to the control group. Statistically significant differences were also found in heart and kidneys absolute weights but as they were observed in pair-fed and high dose groups, they could not be related to test item. No statistically significant changes were observed in absolute organs weights of the satellite males. Statistically significant increases in organ weights relative to bodyweight were observed in the high dose group, probably due to the mean lower BW observed in this group (-25 % of control group). Also, the spleen, thymus, epididymides, seminal vesicles and prostate weights relative to BW were not statistically different from control group, probably because of the same reason, although seminal vesicle and prostate weights showed still lower mean values than control group. Spleen, thymus, epididymides, seminal vesicles and prostate weights were also statistically significantly lower than control group when related to brain weight. As no differences in these organ weights were observed in the recovery animals, these test item-related changes were considered as reversible.
- In females, the only statistically significant differences in mean absolute organ weights were found for liver (increased weight) and ovaries (lower weight than control) in the high dose group. The same significant changes were observed in organ weights relative to brain weight with also a statistically significant lower spleen weight. Like males, many organ weights relative to BW were statistically significantly lower than control group at 3000 ppm, probably due to the lower mean BW observed in this group (-14 % of control group). These potential test item-related changes were reversible as no statistically significant changes in absolute and relative to brain weight organ weights were observed in the 3000 ppm group compared to control group in the satellite animals.

GROSS PATHOLOGY
- No test item-related gross findings were observed at necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Minimal, partially reversible, microscopic changes were observed in testis and epididymides (treated males), liver (females) and kidneys, thymus and spleen (males and females). Changes in testis and epididymis completely disappeared during recovery period. Only partial recovery occurred in satellite males for thymus and spleen. In females, the microscopic changes observed could be considered as reversible and potentially not test item-related as these changes were also observed in control or pair-fed satellite groups.

OTHER
- Spermiology showed statistically significant and dose related lower motility than control group, was found in treated male rats. The difference between pair-fed and control group was small (5.2 %), but statistically significant which showed that feed restriction contributed to the effect. However, as it was more pronounced in the treated groups, a test item effect could not be excluded. Also, statistically significant higher frequencies of all types of spermatozoid abnormalities were observed in the high dose group. All these effects were at least partly reversible.

Effect levels (P0)

Dose descriptor:
LOAEL
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive function (sperm measures)

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

Not applicable

Effect levels (F1)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under these test conditions, no NOAEL (No Observed Adverse Effect Level) could be identified in this study because of the test item related and statistically significant lower sperm motility in the low dose group. The identified LOAEL was therefore 300 ppm.
Executive summary:

In a repeated dose oral toxicity study conducted according to the OECD Guideline 408 and in compliance with GLP, test item, Tetrapropylene benzene was administered daily by feed to groups of Sprague-Dawley rats (10 animals/sex/dose) at dose-levels of 300, 1000 and 3000 ppm in the diet for 13 weeks. A control and pair-fed control groups were included in the study. Three satellite groups (5 animals/sex/group), corresponding to recovery groups, were used: control, pair-fed and 3000 ppm groups. After 90 days of treatment, the test item administration was suppressed and all satellite animals were necropsied at the end of a 28-day treatment-free period. Examinations during the study included: mortality, clinical signs, body weight, food consumption, neurobehavioural examination, ophthalmology, haematology, blood chemistry, urinalysis, spermiology, organ weights, gross pathology and histopathology.

No clinical signs or mortality occurred during the study. Compound-related effect was observed in food consumption in treated animals at 3000 ppm, more pronounced in males than in females. Lower food consumption was associated to the low palatability of the diet containing the test item at 3000 ppm and responsible for the decreased body weight and body weight gain in these groups. No significant changes in food consumption and body weight were noticed at 300 and 1000 ppm. Only statistically significant lower RBC values were seen at doses of 1000 ppm and 3000 ppm in males, which were considered reversible. Lower WBC was observed in treated and pair-fed animals compared to the controls; however, as this effect was found in pair-fed group, it could not be related to test item toxicity. In females, no statistically significant or dose-related findings were observed for all parameters. Only aspartate aminotransferase and triglycerides decreased dose proportionally with statistical significance at 3000 ppm only in males. No clear significant toxicological effect attributable to the test item could be identified from these analyses. No effect of the test item could be identified from the ophthalmology, urinalysis and neurobehavioural examinations. Compound related and reversible effects were observed in the high dose males in spleen, thymus, epididymides, seminal vesicles and prostate absolute weights, with statistically significant decreases recorded. In females, the only statistically significant differences in mean absolute organ weights were found for liver (increased weight) and ovaries (lower weight than control) in the high dose group. Minimal, partially reversible, microscopic changes were observed in testis and epididymides (treated males), liver (females) and kidneys, thymus and spleen (males and females). Changes in testis and epididymis completely disappeared during recovery period. Only partial recovery occurred in satellite males for thymus and spleen. In females, the microscopic changes observed could be considered as reversible and potentially not test item-related as these changes were also observed in control or pair-fed satellite groups. Spermiology showed statistically significant and dose related lower motility than control group. The difference between pair-fed and control group was small (5.2 %), but statistically significant which showed that feed restriction contributed to the effect. However, as it was more pronounced in the treated groups, a test item effect could not be excluded. Also, statistically significant higher frequencies of all types of spermatozoid abnormalities were observed in the high dose group. All these effects were at least partly reversible.

Under these test conditions, no NOAEL (No Observed Adverse Effect Level) could be identified in this study because of the test item related and statistically significant decrease in sperm motility in the low dose group. The LOAEL was 300 ppm.