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Description of key information

In a 4-week oral study on BAB, the NOAEL was 200 mg/kg/day. Effects were either related to local concentrations (forestomach lesions), adaptive, of moderate amplitude/severity, or of types known to be reversible. Such effects were never mentioned for linear alkylbenzenes (apart from growth impairment). Adverse effects on reproductive organs and tissues (mainly in males ; see detail under reproductive toxicity section) were observed down to 400 mg/kg/day included. In a GLP-compliant 90-day repeated dose toxicity study conducted according to OECD guideline 408, a NOAEL of 300 ppm, corresponding to combined test item intake of about 25 mg/kg/day was established, based on microscopic changes in kidneys in males and in liver in females at 1000 ppm. No NOAEL could be identified when considering toxicological effects on male reproductive system (see 7.8.1 section)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
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
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
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: 7-8 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
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral 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.
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
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:
Not applicable
Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
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)
Other examinations:
Spermiology: Sperm motility, morphology, enumeration of homogenization-resistant spermatids and determination of the sperm reserve
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).
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:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
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.
Dose descriptor:
NOAEL
Effect level:
300 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on microscopic changes in kidneys (minimal/mild mineral deposition and vacuolation of tubular epithelium in corticomedullary junction) in males and in liver (minimal hepatocellular vacuolation) in females at 1000 ppm
Critical effects observed:
not specified

None

Conclusions:
Under these test conditions, a NOAEL of 300 ppm, corresponding to combined test item intake of about 25 mg/kg/day, was established based on microscopic changes in kidneys (minimal/mild mineral deposition and vacuolation of tubular epithelium in corticomedullary junction) in males and in liver (minimal hepatocellular vacuolation) in females at 1000 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, a NOAEL of 300 ppm, corresponding to combined test item intake of about 25 mg/kg/day, was established based on microscopic changes in kidneys (minimal/mild mineral deposition and vacuolation of tubular epithelium in corticomedullary junction) in males and in liver (minimal hepatocellular vacuolation) in females at 1000 ppm.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
All studies available are recent GLP-compliant studies conducted according to the most recent OECD guidelines (407 and 408).
System:
urinary
Organ:
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

In a GLP-compliant 90-day repeated dose toxicity study conducted according to OECD guideline 408, a NOAEL of 300 ppm, corresponding to combined test item intake of about 25 mg/kg/day, was established based on microscopic changes in kidneys in males and in liver in females at 1000 ppm. The adverse changes observed at 1000 ppm were not considered as sufficiently severe to consider the classification of the substance as they were limited to minimal or mild microscopic changes in male kidneys (minimal/mild mineral deposition and vacuolation of tubular epithelium in corticomedullary junction) and in females liver (minimal hepatocellular vacuolation) without adverse impact on organ weight, urinalysis and blood chemistry. Toxicological effects on male reproductive system were not considered for the classification of the substance under this hazard class but were taken into account for reproductive toxicity classification.

The substance is not classified for repeated dose toxicity according to CLP Regulation,