Zinc, O,O-mixed (iso-Bu), (iso-Pr), (pentyl) phosphorodithioate

Endpoint summary

Description of key information

oral repeated dose, short term:

source substance Zinc bis(O,O-diisobutyl dithiophosphate) (CAS 68457 -79 -4): GLP, according to OECD Guideline 422, Klimisch 1, rat, oral (gavage), NOEL (portal-of-entry) = 40 mg/kg bw/day (localised injury to the nonglandular portion of the stomach), NOAEL (systemic effects) = 160 mg/kg bw

source substance Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259 -15 -8): GLP, similar to OECD Guideline 407, Klimisch 1, rat, oral (gavage), NOEL (portal-of-entry) = 10 mg/kg bw/day, NOAEL = 125 mg/kg bw/day (systemic effects).

 

Based on the available data and based on the performed studies for CSA as key value the value at which no adverse effect was observed was choosen. This value is a bit higher than the NOEL. Unfortunately the provided data is different, because one study shows the NOEL and the other one shows the NOAEL, but nevertheless the results are in range, because the observed NOAEL of the one substance is higher than the NOEL of the other substance.

 

sub-chronic toxicity: oral

This study was conducted to evaluate the potential toxicity of the substance in Wistar rats following daily administration through oral gavage for 90 consecutive days as per the OECD Guideline 408.

The prepared dose formulations of the test item in the vehicle were administered orally, by gavage, at three graduated dose levels (G2 - low dose, G3 - mid-dose, and G4 - high dose) to male and female Wistar rats for 90 consecutive days. Rats in the concurrent vehicle control group (G1) received vehicle only.

To assess reversibility, persistence, or delayed occurrence of toxic effects, an additional group (G6 – high dose recovery) were treated at the high dose level for 90 days and then observed further for a period of 28 days without any treatment. For comparison purposes, a vehicle control recovery group (G5) was also included and was given vehicle alone over the equivalent period (90 days) and observed further for 28 days.

During the experimental period, all rats were observed for signs of toxicity, morbidity, and mortality. Body weight was recorded at weekly intervals. Body weight change and food consumption were calculated at weekly intervals. An ophthalmological examination was performed before treatment and sacrifices. Neurobehavioral observations were performed prior to treatment and at weekly intervals thereafter. Functional observational battery was performed during 12^th week of the treatment period and last week of recovery period. Vaginal smear was examined on the day of necropsy in all surviving female rats. At the end of the treatment and recovery periods, clinical pathology evaluations were performed on all surviving rats. All surviving rats were sacrificed and subjected to gross pathological examination. The weight of selected organs was recorded for each rat. Microscopic examination of tissues and organs was performed in vehicle control (G1) and high dose (G4) groups.

 

Dose Formulation Analysis: The a.i. concentration and homogeneity results of dose formulation samples collected on days 1, 29, 57, and 85 were within the acceptable range of ±15% of nominal concentration and %CV < 10, respectively.

Mortality and Morbidity: No mortality or morbidity was observed throughout the treatment and recovery periods in male and female rats of control and low dose groups.

Mortality was observed at different interval in rats treated with mid i.e., 150 mg/kg b. wt./day [Male – 2/10; female – 1/10] and high dose i.e., 300 mg/kg b. wt./day [Male – 7/15; female – 2/15] groups. Mortality observed in treatment groups was associated with clinical sign like, gasping, weakness and salivation. Incidence of mortalities was stopped on reduction on doses i.e., 25, 50 and 100 mg/kg b. wt./day from day 37 of male rats and day 36 of female rats. Hence, it was considered as an effect of test item treatment.

Clinical Observations: All rats were normal throughout the dosing period in control and low dose groups. Mild to moderate salivation was observed intermittently during treatment days 6 to 90 in male and female rats of mid and high dose treated groups. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test item rather than an indication of toxicity (Wook-Joon Yu et al, 2011). Therefore, it was considered that the transient salivation observed in this study was of doubtful or no toxicological significance.

Apart from salivation, there was weakness (on days 35 to 37) and gasping (days 62 to 68) also observed in male and female rats of mid and high dose groups. These signs were associated with mortalities. Hence, they were considered as effect of test item treatment and adverse in nature.

Ophthalmological Examination: Ophthalmological examination of all rats did not reveal any abnormalities.

Neurobehavioural Observations: No treatment-related changes were observed in neurobehavioural observations performed in male and female rats from the treatment groups when compared to those respective vehicle control groups.

Functional Observational Battery: No treatment-related changes were observed in functional observational battery parameters performed in male and female rats from treatment groups compared to those respective vehicle control groups.

Body Weight and Body Weight Change: No treatment related changes were observed in the mean body weight and mean body weight change of male and female rats of low and mid dose groups compared to that of vehicle control groups. In high dose groups, significant reduction in body weight and body weight change were observed at different interval in male rats. Body weight gain reduction in male rats of high dose groups was consistent and more than 10% of control groups. It was supported with reduction in food consumption also. Hence, reduction in body weight was considered as an effect of test item and adverse in nature.

Food Consumption: No treatment related changes were observed in the mean food consumption of female rats of treatment groups compared to that of vehicle control group. A significant decrease in food consumption was observed at different interval in male and female rats of low, mid and high dose groups when compared with those respective vehicle control groups. Reduction in food consumption of male rats treated with low and mid dose groups was inconsistent and marginal. Hence, it was considered as an effect of test item but not adverse in nature. Reduction of food consumption at high dose was observed in both sexes and was consistent. In addition, this reduction was observed in more that 10% of control groups and associated with reduction in body weight. Therefore, these changes were considered as effect of test item and adverse in nature.

Hematology and Coagulation: No treatment-related change was observed in hematology and coagulation parameters of rats from treatment groups compared to the respective vehicle control groups.

Clinical Chemistry: No treatment-related change was observed in clinical chemistry parameters of rats from treatment groups compared to the respective vehicle control groups.

Hormone Analysis: No treatment-related change was observed in serum level of T3, T4 and TSH of rats from treatment groups compared to those respective vehicle control groups.

Organ Weight: No treatment-related change was observed in absolute and relative weight of organs in male and female rats of treatment groups compared to those respective vehicle control groups.

Macroscopic Examination: Macroscopic examination did not reveal any treatment related lesion in treatment groups.

Microscopic Examination: Microscopic examination did not reveal any treatment related lesion in treatment groups.

Conclusion:

Initial doses i.e., 150 and 300 mg/kg b. wt./day had produced mortality in male and female rats during dosing days 15 to 36. Additionally, mortality was also noticed after the reduction of doses i.e., 100 mg/kg b. wt./day level (during days 38 to 60). These mortalities were associated with toxic signs like weakness and gasping. Also, the high dose i.e., 300/100 mg/kg b. wt./day was associated with significant reduction in body weight as well as food consumption. All these changes were considered as adverse effects of test item treatment.

Terminal parameters like clinical pathology, organ weight and microscopic examination did not reveal any treatment related adverse changes at any of the dose level.

The No Observed Adverse Effect Level (NOAEL) of the test material was hence concluded as 50 mg/kg b. wt./day under the conditions and procedures followed in this study.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good due to high quality guideline studies

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

Repeated dose toxicity: via oral route - systemic effects

source substance Zinc bis(O,O-diisobutyl dithiophosphate) (CAS 68457 -79 -4)

In a guideline combined repeated dose and reproduction / developmental screening study (OECD 422) conducted according to GLP, WIL Research Labs (2010) evaluated the potential toxic effects of the test substance when administered to rats. This study was designed to evaluate the toxic effects, including neurobehavioral effects, of the test material to parental animals and to evaluate the potential to effect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development. The test material was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats at levels of 10, 40 or 160 mg/kg/day. The low- and mid-dose groups each consisted of 10 rats/sex and the high-dose group consisted of 15 rats/sex. A concurrent control group of 15 rats/sex received the vehicle, mineral oil USP, on a comparable regimen. Ten males/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses. Ten females/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 40-52 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. The extra 5 males and 5 females in the control and high-dose groups were not used for mating and were treated 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.

Test item-related moribundity, clinical findings, and microscopic findings in the non glandular portion of the stomach, characterized by epithelial hyperplasia, hyperkeratosis, and inflammation, were observed in the 160 mg/kg/day group. The injury to the nonglandular portion of the stomach was localized and considered to be irritation from test item portal-of-entry effects. Based on these results, the NOEL for portal-of-entry effects was considered to be 40 mg/kg/day, and excluding the histologic injury to the nonglandular stomach, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 160 mg/kg/day.

 

source substance Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259 -15 -8)

The toxicity potential of the test article, was evaluated in this 28-day study in rats. This study was also conducted to aid in dose level selection for an OECD reproduction/developmental toxicity screening study. The test article in Mazola® Corn Oil was administered orally by gavage to five groups, each consisting of five male and five female Sprague- Dawley Crl:CD® BR rats, for a period of 28 consecutive days. Dosage levels were 10, 50, 125, 250 and 500 mg/kg/day. All animals were dosed at a volume of 5 mL/kg. A concurrent control group of identical design received the vehicle, Mazola® Corn Oil, on a comparable regimen at a dose volume of 5 mL/kg. The animals were observed for clinical signs and effects on body weight, food consumption and organ weights. Necropsies were performed on all animals that died or were euthanized at study termination. A microscopic examination was conducted on selected tissues from the control, 250 and 500 mg/kg/day dose groups.

Three males and one female dosed at 500 mg/kg/day died between study days 6 and 16. These deaths were attributed to treatment. One female dosed at 125 mg/kg/day died on study day 7. The cause of death for this female was unknown. No deaths were observed at the higher dose level of 250 mg/kg/day, and the death of this female was not attributed to oral administration of the test article.

All other animals survived to the scheduled necropsy. Test article related clinical signs included changes in fecal consistency, discoloration of the feces, tan, yellow or brown staining on various body surfaces, rales, salivation and aggression in the 125, 250 and 500 mg/kg/day group males and females. Rales and salivation were also observed in the 50 mg/kg/day group males. Body weight gain was inhibited in the 250 mg/kg/day group males and the 500 mg/kg/day group males and females during the first two days of dosing. Inhibition of body weight gain continued in the high dose group males through study day 12. Food consumption was slightly reduced in the 500 mg/kg/day group males and females during the first week of dosing.

At the scheduled necropsy, thickened mucosa in the nonglandular portion of the stomach was observed in one 500 mg/kg/day group male and in one and two females in the 250 and 500 mg/kg/day groups, respectively. At the microscopic examination, one 250 mg/kg/day group male and all 500 mg/kg/day group females had submucosal edema in the glandular andlor non-glandular portions of the stomach. Three of the high dose group females also had suppurative inflammation, primarily in the non- glandular portion of the stomach. These findings are consistent with a response to a gastric irritant. Mean absolute and relative adrenal gland weights in the 250 and 500 mg/kg/day group males and females were increased when compared to the control group values. No test article related histopathological lesions were observed in the adrenal glands to account for the increases.

Based on the data obtained, a dose level of 500 mg/kg/day would exceed a maximum tolerated dose for a subsequent reproduction/developmental toxicity study in rats. Treatment-related increases in mean absolute and relative adrenal gland weights occurred at the 250 and 500 mg/kg/day dose levels. However, no test article-related histopathological lesions were observed in the adrenal glands to account for the increases. Based on these findings, the No Adverse Effect Level NOAEL was determined to be 125 mg/kg/day. A dose level of 10 mg/kg/day was considered to be the NOAEL (no observable adverse effect level) for local effects.

 

sub-chronic toxicity: oral

This study was conducted to evaluate the potential toxicity of the substance in Wistar rats following daily administration through oral gavage for 90 consecutive days as per the OECD Guideline 408.

The prepared dose formulations of the test item in the vehicle were administered orally, by gavage, at three graduated dose levels (G2 - low dose, G3 - mid-dose, and G4 - high dose) to male and female Wistar rats for 90 consecutive days. Rats in the concurrent vehicle control group (G1) received vehicle only. To assess reversibility, persistence, or delayed occurrence of toxic effects, an additional group (G6 – high dose recovery) were treated at the high dose level for 90 days and then observed further for a period of 28 days without any treatment. For comparison purposes, a vehicle control recovery group (G5) was also included and was given vehicle alone over the equivalent period (90 days) and observed further for 28 days. 

Initial doses i.e., 150 and 300 mg/kg b. wt./day had produced mortality in male and female rats during dosing days 15 to 36. Additionally, mortality was also noticed after the reduction of doses i.e., 100 mg/kg b. wt./day level (during days 38 to 60). These mortalities were associated with toxic signs like weakness and gasping. Also, the high dose i.e., 300/100 mg/kg b. wt./day was associated with significant reduction in body weight as well as food consumption. All these changes were considered as adverse effects of test item treatment.

Terminal parameters like clinical pathology, organ weight and microscopic examination did not reveal any treatment related adverse changes at any of the dose level.

The No Observed Adverse Effect Level (NOAEL) of the test material was hence concluded as 50 mg/kg b. wt./day under the conditions and procedures followed in this study. The LOAEL it is thus extrapolated to be 100 mg/kg b. wt./day. No target organ was identified in the study.

Justification for classification or non-classification

As per the guidance values (C) of CLP Regulation the substances can be classified in STOT-RE 1 or STOT-RE 2.

In the case of the substance, the study on the substance, the LOAEL was identified as 100 mg/kg bw/day, and thus a classification as a STOT-RE 2 should be attributed. However, this dose was not given for the whole duration of the study (90 days), but as for day 37. The animals in this group were previously dosed at 300 mg/kg bw/day. Therefore the LOAEL as 100 mg/kg bw/day is actually calculated in prorated doses in male/female as 180/178 mg/kg bw/day. Furthermore, no target organ was identified in the specific study. The NOAEL and consequently the LOAEL was defined due to adverse effects in body weight, food consumption, morbidity/mortality and clinical signs such as weakness and gasping.

For this reasons, as per the CLP Regulation (EC) No 1272/2008 classification of this substance is not required for prolonged exposure.