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Description of key information

The test item caused adverse effects on kidneys upon subchronic feed application to rats with a NOAEL of 1500 ppm (ca 75 mg/kg bw). Upon gavage dosage, rats do not suffer from adverse findings at doses of up to 1000 mg/kg bw as shown in a GLP-compliant 28d study and in a GLP-compliant OECD 422 study. In dogs, liver was identified as target organ in a two year feeding study; the NOAEL was 3000 ppm (ca 100 mg/kg bw).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subchronic
Species:
rat
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

Results of repeated dose toxicity are dependent on whether gavage or feed application is used and what species is tested. Kidney is identified as the most sensitive organ in rats upon feed application with a NOEL of 1500 ppm (ca. 75 mg/kg bw). No effects are observed upon subacute gavage application of 1000 mg/kg bw in a GLP-compliant OECD 407 study an in a enhanced GLP-compliant OECD 422 study. The feeding studies were performed between 1965 and 1975 and have limitations in the number of investigated parameter. In dogs, liver was identified as the target organ upon chronic feeding. As the target organs kidney and liver are sufficiently investigated and as no other target organs were identified in the two state of the art gavage studies, the database is considered sufficient and acceptable for hazard assessment. Detailed description is given in the following paragraphs.

In a modified GLP-compliant OECD 422 study, the test substance Octabenzone was administered daily as an aqueous preparation to groups of 12 male and 12 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals (12 male and 12 female Wistar rats) were dosed daily with the vehicle only (0.5% Carboxymethylcellulose suspension in drinking water + 5 mg/100 mL Tween 80). The duration of treatment covered a 10 weeks pre-mating period in the males, 2 weeks pre-mating in the females, 2 weeks mating period in both sexes, approximately 2 days post-mating in males, and the entire gestation period as well as approximately 30 days of the lactation period (21 days lactation and up to 9 days post-weaning) and 35 days post-mating (for sperm negative females). Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was demonstrated over a period of 7 days under ambient conditions. No clinical findings were caused by the test substance in the parental animals F0 up to the limit dose of 1000 mg/kg bw/d. Food consumption and body weight (change) of the parental animals showed no treatment-related, adverse changes. In the subsequent investigations including the detailed clinical observation (DCO), the functional observational battery (FOB) and measurement of motor activity (MA) no treatment related, adverse differences to control were observed at any dose level. Regarding clinical pathology, no treatment-related, adverse effects were observed up to 1000 mg/kg bw/d. At PND 13 in male pups of test groups 13 (1000 mg/kg bw/d) T4 levels were significantly lower compared to controls. However, no histopathological correlate or any alteration of the thyroids was found. Therefore, the statistically significant decrease of T4 in male PND 13 pups of test group 13 was regarded as treatment-related but not adverse. Regarding pathology, there were neither treatment-related organ weight changes nor gross lesions. Histopathology of the thyroid glands in F0 parental male animals and PND 13 male pups did not show any relevant morphological changes consistent with functional alterations. All histopathological changes occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity of Octabenzone was 1000 mg/kg bw/d.

The test article was further studied for oral toxicity in rats in a GLP-compliant 28-day repeated dose toxicity test at doses of 0, 20, 140, and 1000 mg/kg/day applied by gavage (MHLW, 1995), following OECD testing guideline 407 including a 14-day treatment free recovery period. Each group consisted of each 6 of males and females, and the control group was given only solvent (0.5% CMC-Na aqueous solution with 0.1% Tween 80 added). Control and high dose groups consisted of additional 6 animals per sex for recovery investigations. In this study, no mortalities occurred and no test substance-related changes were noted in terms of clinical observation, body weights, food consumption, and the findings obtained from hematology tests, blood chemical examination, urinalysis and pathological examination. The NOEL for the subacute gavage study is considered to be 1000 mg/kg/day for both sexes.

In a 90-day feeding study in rats with 0.2, 2 and 5% in the diet, a NOAEL was not identified (TNO, 1967). The study design showed several deviations from OECD guideline 408 (1998): Biochemical analyses and ophthalmologic examinations were not performed, individual details of macroscopic findings and individual body weights were not reported, no sensory reaction to stimuli test was performed, and food consumption was not measured weekly. The study pre-dates GLP requirements. Deaths occurred in the group administered the highest dose (5.0%). Growth rate, food consumption and food efficiency were decreased at the two highest dosage levels in males and females. At the 0.2 % feeding level, only females showed consistently, though not significantly, lower body weights than the controls. Hemoglobin content, packed cell volume and number of erythrocytes were decreased at the two highest dosage levels in males and females. The relative weights of kidneys and livers were increased at feeding levels of 2.0 and 5.0% in both sexes. Kidney weight was slightly increased also at the lowest feeding level. Adrenal weights were increased at all feeding levels in females but not in males. No changes of toxicological significance were noted during histopathological examinations of the adrenals. Thyroid weights were higher than in controls in all feeding levels. However, these changes did not display a proper dose response relationship and furthermore, the level of significance was only reached in the mid dose group in males. Since there were no correlating findings during histological examinations, the toxicological significance is doubtful and this finding is considered to be non-adverse. At autopsy enlarged, discolored livers, palish-green kidneys, and uroliths in the renal pelvis and in the urinary bladder were observed at the two highest dosage levels, both in males and females. Upon microscopy signs of toxic tubular nephrosis were observed in kidneys at all feeding levels and hyperplastic epithelium in urinary bladders at the two highest feeding levels. Hemoglobin content, packed cell volume and number of erythrocytes were decreased at the two highest dosage levels in males and females. Glucose 6-phosphatase activity in the livers was increased at 0.2% and above, whereas in the kidneys the activity of this enzyme was not distinctly affected at any of the feeding levels. As the NOAEL was not clearly identified, the study was repeated a year later.

In the repeat study the same general setup was used at dose levels of 0.065, 0.1, or 0.15 % with groups of ten male and ten female young albino rats (TNO, 1968). Observations were made of general appearance and behavior, growth, food intake and efficiency, and of a number of hematological factors. Examinations were carried out terminally on the blood and the liver for various enzyme levels. At week 14 all rats were examined grossly and the weights of ten different organs were recorded. Extensive histopathological examinations were carried out. No deaths or untoward reactions were recorded. Gain in body weight, food intake, food efficiency, hematology, glutamic-pyruvic transaminase, glutamicoxaloacetic transaminase and alkaline phosphatase in the serum showed no treatment related abnormalities at any dietary level. The relatively low white blood cell counts which occurred in males at 0.15 and 0.10 % were within the range of normal values in our strain of rats. Moreover, in the previous study with the same test compound fed at much higher levels no indications of leucopenia were observed. Therefore, no toxicological signicance is attached to the low leucocyte counts at 0.15 and 0.10 %. Organ-to-body weight ratios revealed slightly increased kidney ratios at the highest feeding level in females, and slightly increased thyroid ratios at the two highest levels in males. These effects were not accompanied by histological changes, therefore these elevated organ weights were considered to be not adverse. Gross and microscopic examination did not reveal pathological changes attributable to the feeding of the test item. The principal toxic effect induced by high dietary levels in the previous study consisted of toxic tubular nephrosis, which occurred to a slight extent also at the lowest feeding level of 0.2 %. In the present study, this abnormality was not observed at all at 0.15 %. Therefore, it is concluded that 0.15 % was a no-toxic-effect level in the present experiment.

Another subchronic feeding study is available with rats. This study was conducted to determine the potential oral toxicity of the test article upon continuous administration in feed for 3 months to rats (965). The study was based on a 30-day feeding study in which at 2.5% and 5% animals had poor appearance, partly hematurea, yellow masses in renal tubules and bladder found upon microscopy. In the subchronic study, determination of test substance stability, urinalysis, weight of all organs, clinical biochemistry and some other parameters were not included in the study design. The test material was fed at dietary levels of 0, 0.2, 0.6, or 1.8% to groups consisting of 20 males and 20 females. The diet was prepared at weekly intervals and was available ad libitum. The body weight of each rat was recorded twice a week. The food consumption of each group was determined weekly. Hematological data were collected from 5 animals of each sex animals in the 11th week. At the time of autopsy, liver and kidney weights were determined for 10 animals of each sex/group. 15 animals of each sex/group were examined grossly, weights of organs were recorded, and extensive histopathological examinations were conducted. The overall appearance and behavior of all animals were good throughout the feeding period. Two deaths occurred during the study period, a 0.2% female found dead on day 5 and a 0.6% male which lost weight in week 11 and died in week 12. Advanced autolysis prevented the determination of the cause of death in both instances. However, since no deaths occurred at the maximum feeding level of 1.8% it was concluded that the deaths were not related to the test article. There were no significant differences in mean weight gain between any test group and its corresponding control. There were no significant differences in mean food intake between test and control animals. There were no significant differences in mean hemoglobin or mean hematocrit between and of the test groups. Females in the 0.2% group had a very slight but significant decrease in total leukocyte, but this was judged to be coincidental and unrelated to feeding of the test material as not differences in leukocyte counts were noted in the two higher dietary levels. Mean liver weight of the 0.2% males and of both sexes at 0.6% was significantly increased, but no effect on liver weight was noted in the 1.8% dietary groups. Thus, this was determined to be unrelated to the test article. Mean kidney weight was significantly decreased for the 0.2% females and significantly increased for the 1.8% males. There was a slight, but not significant increase in the kidney weights of the 1.8% females. The observed increases in kidney weight at the 1.8% dietary levels could be test article related. No significant lesions were encountered at the time of autopsy. The no-toxic-effect level (NOEL) was 0.6% in the diet. As urinalysis was not part of the above study, the NOAEL is not taken into account for hazard assessment.

Additional investigations on hematurea in rats were performed. In the first study, doses of 6000 and 18000 ppm were fed for 20, 28 or 35 days. Hematurea, proteinurea and crystallurea were observed only in treated animals and not in control animals. As a follow-up study, a purified sample and the original test substance were applied in the feed at concentrations of 2000, 6000 and 18000 ppm to each four male rats for 21 days. This time, occult blood, hematurea, proteinurea and crystallurea were observed both in control group animals and in treatment group animal. The incidence increase was increased at 18000 ppm and appeared to be in the range of the control group at the other dose groups. The findings were confirmed in the third study.

Subchronic toxicity was also investigated in a dietary feeding study in dogs (1965). There were only 2 animals per sex and dose group, and no analysis of feed was performed. Some parameters were not assessed (eg no urinalysis, no neurobehavioral examination). Feeding was done over 125 – 127 days. The test substance was initially fed at dietary levels of 0, 2000, 6000, or 18000 ppm. Due to unpalatability of the 18000 ppm dietary level, it was reduced to 4000 ppm at the end of week 2. After 124-127 days on test, animals were anesthetized and exsanguinated from the left brachial artery. Spleen, pancreas, liver, adrenal, kidney, thyroid, heart, pituitary, brain and testes were weighed for all dogs. Alkaline phosphatase, glucose and urea nitrogen were determined in plasma. In addition, BSP (bromosulfopthalein) clearance was determined on each of the 16 dogs prior to the start and at week 15. At the start and after 14 weeks on test, hemoglobin, hematocrit, and total leukocyte counts were determined. The general appearance and behavior of all dogs were normal during the test period. While there were differences in the percentage of gain between animals in the various groups, there was no evident correlation between weight gain and either food consumption or dietary level of the test item. No findings related to treatment were reported during hematology and clinical chemistry. All individual values for plasma glucose, urea nitrogen and percent retention of BSP were within acceptable ranges, and there were no significant differences in the mean values of the groups for a given measurement. the weights of each organ were generally comparable for each group and within ranges previously encountered in this laboratory. There were no gross findings at autopsy that could be considered related to the feeding of the test item. There was only the customary collection of items of incidental pathology that would be encountered in the examination of any group of animals selected at random. The only commonly occurring change was a mild, verrucous endocarditis of the right atrio-ventricular valves. This is a common spontaneous degenerative lesion in dogs, and is asymptomatic. It was quite mild and was seen at all levels, including controls. Sections of muscle, rib, spleen, mesenteric lymph node, pancreas, liver and gall bladder, stomach, ileum, caeci, colon, urinary bladder, gonads, uterus or prostate, adrenal, kidney, thyroid and parathyroid, lung, heart, pituitary, and brain were processed for microscopic examination. None of the lesions encountered microscopically appear to be related to feeding of the test article. It is concluded that feeding of the test item to dogs at levels as high as 6000 ppm for 120 days did not result in either gross or microscopic lesions referable to ingestion of the product. The NOAEL was therefore identified as 6000 ppm.

 

A two-year dietary feeding study in dogs was performed in 1969 (Cyanamid 1969). The study has the following deficits: No urinalysis, no analysis of feed, organ weight determination only for liver and kidney, no histopathology of eyes, spinal cord and nerves. The substance was incorporated into the diet to give concentrations of 1000 ppm, 3000 ppm, and 9000 ppm. Fresh diets were prepared at weekly intervals. From the offered amount of food, an average daily intake of the test item is estimated as 33, 100 and 300 mg/kg bw. Intracellular brown pigment was noted in the cortical tubules in the kidneys from the control as well as from the 3000-ppm and 9000-ppm animals. The cells of the collecting tubules in the medullary ray of the kidneys of females in the control and test groups were more vacuolated than were the corresponding structures in the males. The NOAEL was identified at 3000 ppm. Higher doses caused adverse effects on liver observed for females (3-fold increase in serum ALP and moderate centriluobular mixed inflammatory cell infiltration) at 9000 ppm after 24 months. Slight effects on BUN and ALP were observed in males.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No serious irreversible effects were observed at doses of less than 100 mg/kg bw upon subchronic application. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.