Registration Dossier

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

The key study was performed according to OECD 422: A Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Rats, with Recovery.

The test item in the vehicle (corn) was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and 2 mid-dosage groups (Groups 2, 3, and 4, respectively) each consisted of 12 males and 12 females, and the high-dosage

group (Group 5) consisted of 17 males and females. Dosage levels were 100, 200, 500, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group (Group 1) of 17 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 10 weeks of age at the beginning of test item administration. Twelve males/group selected for pairing received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 31 doses. Twelve females/group selected for pairing received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-51 doses; however, these females should also have been dosed on lactation day 4 but were not administered the test item. The females that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-

cohabitation day 25) for a total of 39-52 doses. The extra 5 rats/sex in the control and high-dose groups were not used for mating but were treated on a comparable regimen beginning on study day 0; following 28 doses for the males and 40 doses for the females, these animals were assigned to the post-treatment period and remained on study for a 14-day non-dosing period.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and locomotor activity data were recorded for 6 males/group following approximately 28 days of dose administration and for 6 females/group on lactation day 4. All F0 females were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (hematology, serum chemistry, and urinalysis) were performed on 6 F0 animals/sex/group at the treatment period necropsy and on all animals at the post-treatment period necropsy. The F0 males were euthanized following completion of the mating period, and the F0 females were euthanized on lactation day 5. The 5 rats/sex in the control and high-dosage groups assigned to the post-treatment period were euthanized following the 14-day non-dosing period.

Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dosage groups at the treatment period necropsy. In addition, the liver and thyroid glands from all animals in the low- and mid-dosage groups and all animals in the post-treatment phase were examined microscopically.

One male each in the 500 and 1000 mg/kg/day groups were found dead on study days 31 and 8, respectively. The male in the 1000 mg/kg/day group lost 114 g of body weight and had an unkempt appearance, red material on the urogenital area, forelimbs, ventral abdomen, nose and mouth, and decreased defecation prior to death. Findings for the 500 mg/kg/day group male were limited to red material around the mouth 1 week prior to death. These deaths were attributed to the test item. One female each in the 500 and 1000 mg/kg/day groups were euthanized in extremis due to test item-related dystocia on gestation day 22 and lactation day 0, respectively. Microscopic findings for these females included test item-related coagulative necrosis of the liver; this finding was considered the cause of moribundity. All other animals survived to the scheduled necropsies. Test item-related clinical findings noted in the 100, 200, 500, and 1000 mg/kg/day group males and females consisted of salivation prior to dose administration or clear material around the mouth and/or nose. These findings were attributed to irritating properties of the test item and were not considered adverse. Other test item-related clinical findings noted in the 200, 500, and 1000 mg/kg/day groups consisted of red material around the mouth. In the 1000 mg/kg/day group males and females, yellow material on the urogenital area, red material around the nose, and soft stool were also noted. These findings were considered adverse. Mean body weight losses and/or lower mean body weight gain and food consumption (generally statistically significant) were observed in the 500 and 1000 mg/kg/day group males generally throughout the treatment period. As a result, mean body weights in these males were statistically significantly lower (9.2% and 13.4%, respectively) than the control group on study day 30. Mean body weights and body weight gain in the 1000 mg/kg/day during the post-treatment period remained lower than the control; however, mean body weight gain was improving slightly. Lower mean food consumption throughout gestation and lower mean body weight gains late in gestation (days 14-20) were noted in the 1000 mg/kg/day group females, resulting in a mean body weight that was 5.8% lower than the control group on gestation day 20; some of the differences were statistically significant. The effects on mean body weight gain during gestation in the 1000 mg/kg/day group females was attributed to the test item-related lower mean number of pups and an increase in the mean number of unaccounted-for sites, not to maternal toxicity. No test item-related effects on mean body weights or body weight gains were observed in the 1000 mg/kg/day group females during the post-treatment period. Test item-related clinical pathology findings (occasionally statistically significant) in male rats included the following. Longer mean prothrombin and activated partial thromboplastin times and higher mean urine volumes and lower urine osmolality were noted at > 100 mg/kg/day. Minimally to slightly higher mean albumin, total protein, and alkaline phosphatase levels were noted at 500 and 1000 mg/kg/day. Slightly lower mean erythrocyte and eosinophil counts, alanine aminotransferase, triglyceride, chloride, phosphorus, and potassium levels and slightly higher mean reticulocyte counts and red blood cell distribution width, mean globulin, total bilirubin, creatinine, and cholesterol, were noted at 1000 mg/kg/day. Test item-related findings were considered to be adverse for prothrombin > 200 mg/kg/day and for activated partial thromboplastin time at 1000 mg/kg/day. These findings resolved in the 1000 mg/kg/day group males during the recovery period with the exception of slightly lower mean erythrocyte and eosinophil counts. Nonadverse, test item-related findings (occasionally statistically significant) in female rats consisted of minimally to slightly mean higher albumin levels at > 100 mg/kg/day, higher mean cholesterol levels and lower mean alanine transferase levels at > 200 mg/kg/day, lower erythrocyte counts, and mean hemoglobin and hematocrit levels and urine osmolality and higher mean total bilirubin levels at > 500 mg/kg/day, and slightly higher mean red blood cell distribution width, and alkaline phosphatase levels, and slightly lower mean triglyceride levels at 1000 mg/kg/day. Following the post-treatment period, mean percent and absolute reticulocyte counts were slightly lower than the control group in the 1000 mg/kg/day group females. The mean hemoglobin distribution width in the 1000 mg/kg/day group females was slightly lower than controls on study day 54 and was considered to be secondary to the lower mean reticulocyte counts. No test item-related effects were noted on male and female mating and fertility, male copulation, or female conception indices, or gestation lengths at any dosage level. One and 2 females in the 500 and 1000 mg/kg/day groups, respectively, were noted with dystocia, resulting in the euthanasia of the 500 mg/kg/day group female and 1 of the 1000 mg/kg/day group females. Dystocia was attributed to the test item. Findings for these females that were euthanized included active vaginal hemorrhage, weakness, pallor, body cool/cold to the touch, hypoactivity, and torpid/tense abdomen. The female in the 500 mg/kg/day did not deliver any pups; however, dead fetuses and an early resorption in utero. The female in the 1000 mg/kg/day group that was euthanized delivered 3 pups, but left them unattended; this female also had dead fetuses and an early resorption in utero. The other female in the 1000 mg/kg/day group noted with dystocia was pale and cool to the touch on lactation day 2 and was euthanized with total litter loss on that day (after delivering 13 pups on lactation day 0). A statistically significantly higher mean number of unaccounted-for sites was noted in the 1000 mg/kg/day group females compared to the control group. A lower mean number of F1 pups born and live litter size on PND 0 were noted in the 1000 mg/kg/day group; the differences from the control group were not statistically significant. The lower mean number of pups born corresponded in part with the higher mean number of unaccounted-for sites observed in the F0 females in this group. Statistically significantly lower mean postnatal survival noted in the 1000 mg/kg/day from birth to PND 4 group was attributed to the test item. No test item-related macroscopic findings were noted at the necropsy of F0 animals that were found dead, euthanized in extremis, or at the scheduled necropsies.

Test item-related effects on organ weights consisted of lower mean thymus weights in the 200, 500, and 1000 mg/kg/day group males, lower mean spleen, and heart weights in the 500 and 1000 mg/kg/day group, lower mean brain weight in the 1000 mg/kg/day group males, higher mean liver weights in the 500 and 1000 mg/kg/day group males, and higher thyroid/parathyroid weights in 200, 500, and 1000 mg/kg/day group males and females. Some of the differences from the control group were statistically significant. Mean liver and thyroid gland weights remained higher (generally statistically significant) in the 1000 mg/kg/day group females at the post-treatment necropsy. Corresponding test item-related microscopic findings consisted of midzonal hepatocellular vacuolation, hepatocellular cytomegaly in the 200, 500, and/or 1000 mg/kg/day group males and females and thyroid follicular cell hyperplasia in the 200, 500, and 1000 mg/kg/day group males and females. The differences from the control group were generally statistically significant. The severity of hepatocellular vacuolation and thyroid follicular cell hyperplasia in the 1000 mg/kg/day group males and females was less at the post-treatment period necropsy; however, the differences from the control group generally remained statistically significant. There was no correlation between these microscopic findings and the clinical pathology results.

Under the conditions of this screening study, no effects on F0 reproductive performance were noted at any dosage level. However, dystocia was present at ≥500 mg/kg/day, and a higher mean number of unaccounted-for sites were noted at 1000 mg/kg/day. Therefore, a dosage level of 200 mg/kg/day was considered to be the no-observed-effect level (NOEL) for reproductive toxicity of the test material when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for neonatal toxicity was considered to be 500 mg/kg/day based on lower mean number of viable pups on PND 0 and postnatal survival through PND 4 in the 1000 mg/kg/day groups.

An expert review of the data from the OECD 422 screening study with repeat oral dosing for 28-days indicates that the effects observed at 200 mg/kg/day are adaptive with animals in the recovery groups showing a reduction in symptoms. The NOAEL of 100 mg/kg bw/day as suggested by the Study Director is based on minor and reversible effects on organ weights and microscopic findings at 200 mg/kg bw/day whereas more significant toxic effects were noted at 500 and 1000 mg/kg bw/day. Consequently it is considered more appropriate to propose a LOAEL of 500 mg/kg bw/day, a NOAEL of 200 mg/kg bw/day and 100 mg/kg bw/day as the NOEL for systemic toxicity.

Short description of key information:
Under the conditions of this screening study, no effects on F0 reproductive performance were noted at any dosage level. However, dystocia was present at ≥500 mg/kg/day, and a higher mean number of unaccounted-for sites were noted at 1000 mg/kg/day. Therefore, a dosage level of 200 mg/kg/day was considered to be the no-observed-effect level (NOEL) for reproductive toxicity of the test material when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for neonatal toxicity was considered to be 500 mg/kg/day based on lower mean number of viable pups on PND 0 and postnatal survival through PND 4 in the 1000 mg/kg/day groups.
An expert review of the data from the OECD 422 screening study with repeat oral dosing for 28-days indicates that the effects observed at 200 mg/kg/day are adaptive with animals in the recovery groups showing a reduction in symptoms. The NOAEL of 100 mg/kg bw/day as suggested by the Study Director is based on minor and reversible effects on organ weights and microscopic findings at 200 mg/kg bw/day whereas more significant toxic effects were noted at 500 and 1000 mg/kg bw/day. Consequently it is considered more appropriate to propose a LOAEL of 500 mg/kg bw/day, a NOAEL of 200 mg/kg bw/day and 100 mg/kg bw/day as the NOEL for systemic toxicity.

Under the conditions of this screening study, no effects on F0 reproductive performance were noted at any dosage level. However, dystocia was present at ≥500 mg/kg/day, and a higher mean number of unaccounted-for sites were noted at 1000 mg/kg/day. Therefore, a dosage level of 200 mg/kg/day was considered to be the no-observed-effect level (NOEL) for reproductive toxicity of the test material when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for neonatal toxicity was considered to be 500 mg/kg/day based on lower mean number of viable pups on PND 0 and postnatal survival through PND 4 in the 1000 mg/kg/day groups.
An expert review of the data from the OECD 422 screening study with repeat oral dosing for 28-days indicates that the effects observed at 200 mg/kg/day are adaptive with animals in the recovery groups showing a reduction in symptoms. The NOAEL of 100 mg/kg bw/day as suggested by the Study Director is based on minor and reversible effects on organ weights and microscopic findings at 200 mg/kg bw/day whereas more significant toxic effects were noted at 500 and 1000 mg/kg bw/day. Consequently it is considered more appropriate to propose a LOAEL of 500 mg/kg bw/day, a NOAEL of 200 mg/kg bw/day and 100 mg/kg bw/day as the NOEL for systemic toxicity.

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL