Registration Dossier

Administrative data

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

experimental study planned (based on read-across)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
Please refer also to the read-across statement attached in section 13

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The target and the source substances are structurally similar substances that share the common organometallic core structure consisting of a central zinc metal bonded to four alkyldithiophosphate esters (ligands) by coordinate covalent bonds -Zn[(S2P(OR)2]2. Structural variations between the target and the source substances are related only to the alkyl (R) groups of the alkyldithiophosphate ligands. The substances in this category give thus rise to an (identical) common compound Phosphorodithioic acid moiety that can be released by the breakage of ester bonds and dissociation from the Zinc complex to which the organism would be exposed if the target substance was tested in the toxicity studies. Exposure to the parent compounds (non-transformed constituents) and to the counter alkyl alcohols, possibly released by hydrolysis of P-O bonds – non-common compounds – would not influence the prediction of the (eco)toxicological properties because they are considered to have the same biological targets and to cause the same type of effects through a common underlying mechanism due to the same functional groups (zinc cation, phosphorodithioic cation and aliphatic alcohol anionic moieties). The impurities of the target and the source substances are not expected to impact the prediction because they are identical or, if slightly structural different, belong to the same class of compounds with the same functional groups and their percentages are very low.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
The results of the two source substances indicate that local irritation of the gastrointestinal tract is the primary effect of the substances in the ZDDP category. This is also in line with the skin and eye irritating properties of the substances.
Since the main constituents of the target substance are structurally similar to the constituents of the source substances with the same functional groups and the alkyl chain lengths of phosphoroditioate moieties are in the range of the established ZDDP category (C3-C12), the same mode of toxicological action is expected for the target and the source substances. The constituents of the target substance do not possess functional groups associated with other mode of actions or toxicity effects. Toxicokinetic behavior of the constituents of the target substance is expected to be essentially the same as that of the source substance. Based on the results of the oral repeated dose toxicity studies with the source substances and other ZDDP category members, it is evident that the structural dissimilarities – the chain lengths of the alkyl rests – did not result in different strength of the toxicity effects. Even if absorption through GI tract of the category members with shorter alkyl rests and theoretically higher absorption rates (due to the increased water solubility and decreased molecular weights favouring absorption) is higher, their intrinsic properties are essentially the same as the findings in the toxicity studies are very similar. The impurities of the target substance are considered not to contribute to the toxicity effects because they are also structurally similar to the impurities of the source substances and consist of substances of simple structure without specific mode of action. Furthermore, their amounts are very low. Therefore, it is predicted that the target substance would possess the same toxicity potential by repeated oral exposure as the source substance.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
not specified
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
not specified
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
Three males in the 500 mg/kg/day group died, one 500 mg/kg/day group female died. The most remarkable clinical signs observed prior to death in these animals consisted of hypoactivity, body cool to touch, hunched appearance, unkempt appearance, extremities pale in color and/or respiratory distress (rales, labored respiration, shallow respiration). One female in the 125 mg/kg/day group died. This female was noted to have the oral cavity obstructed by a thick brown material on the day of death. Other remarkable clinical signs for this female prior to death included hypoactivity, body cool to the touch, hunched appearance, unkempt appearance, extremities pale in color, excessive chewing and respiratory distress (rales, labored respiration, shallow respiration).
All other animals survived to the scheduled necropsy. The predominant clinical signs, observed at a similar incidence and severity throughout the study, consisted of the following. Changes in fecal consistency (soft stool, diarrhea, mucoid diarrhea, mucoid feces), discoloration of the feces and tan, yellow or brown staining on various body surfaces were observed in the 125,250 and 500 mg/kg/day group males and females, primarily at the daily examinations prior to dosing. Rales occurred in the 50 mg/kg/day group males and the 125, 250 and 500 mg/kg/day group males and females, primarily at the daily examinations and one hour following dosing. Salivation was observed in these same dose groups at the time of dosing. Aggression was noted in the 125, 250 and 500 mg/kg/day group males and females at the time of dosing.

Other clinical findings in the treated groups were those commonly observed in laboratory rats andlor were limited in occurrence. These findings generally occurred in a non dose related manner or at a similar incidence in the control group.

Mortality:
Three males in the 500 mg/kg/day group died, one each on study days 6, 8 and 14 (weeks 0, 1 and 2, respectively). One 500 mg/kg/day group female died on study day 16 (week 2).
One female in the 125 mglkglday group died on study day 7 (week 1).

BODY WEIGHT AND WEIGHT GAIN
Mean body weight losses occurred in the 500 mg/kg/day group males and females during the initial two days of dosing (days 0-2). The differences from the control group were statistically significant (p <0.01). Mean body weight losses and reduced mean body weight gains were observed in the 500 mg/kg/day group males during study days 2-4 through 10-12. The mean body weight loss during study days 10-12 was statistically significant (p<0.05) when compared to the control group. Mean body weight gains in the 500 mg/kg/day group females were similar to the control group values during these intervals. Throughout the remainder of the study (days 12- 14 through 26-28), mean body weight changes in the 500 mg/kg/day group males and females were unaffected by test article administration; no trends were apparent. For the overall study period (days 0-28), the 500 mg/kg/day group males had a reduced mean body weight gain (not statistically significant), while the females in this group showed an increased mean body weight gain (p<0.05) when compared to the control group. Mean body weights in this group were lower than the control group values for days 2 through 12 in the males and for days 2 and 4 in the females. The differences between the control and 500 mg/kg/day group females on days 2 and 4 were statistically significant (p<0.01 and p<0.05, respectively).

At a dose level of 250 mg/kg/day, a slightly reduced mean body weight gain occurred in the males during days 0-2. No other adverse effects on mean body weight changes and no differences in mean body weights were apparent in the 250 mg/kg/day group when compared to the control group.
Mean body weights and body weight changes in the 10, 50 and 125 mg/kg/day group males and females were unaffected by test article administration.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption, evaluated as g/animal/day, was slightly reduced in the 500 mg/kg/day group males and females during week 0-1. The differences from the control group were not statistically significant. Throughout the remainder of the study (weeks 1-2 through 3-4), food consumption in the 500 mg/kg/day group males and females was slightly higher than that in the control group. The differences between the control and 500 mg/kg/day group females during weeks 1-2 and 3-4 were statistically significant (p<0.05 and p<0.01, respectively).
Food consumption in the 10, 50, 125 and 250 mg/kg/day group males and females was similar to the control group throughout the study.

FOOD EFFICIENCY
No data.

WATER CONSUMPTION
No data.

OPHTHALMOSCOPIC EXAMINATION:
No data.

HAEMATOLOGY
No data.

CLINICAL CHEMISTRY
No data.

URINALYSIS
No data.

NEUROBEHAVIOUR
No data.

ORGAN WEIGHTS
Mean absolute adrenal gland weights were increased by 27 % and 5 1% for males and 20% and 18% for females in the 250 and 500 mg/kg/day groups, respectively. The differences between the control group and the 250 and 500 mg/kg/day group males were statistically significant (p<0.05 and p<0.01, respectively). Mean adrenal gland weights relative to final body weight ratios and mean adrenal gland weights relative to brain weight ratios in these groups were also increased when compared to the control group values. All of the male differences and the relative to brain weight ratio differences in the females were statistically significant (p<0.05 or p <0.01). Mean absolute and relative adrenal gland weights in the 10, 50 and 125 mg/kg/day groups were similar to the control group values.
No other test article related effects on organ weight data were observed at any dose level. Increases were observed in mean absolute and relative (to final body weight) testes (p<0.05) and epididymides weights in the two surviving 500 mg/kg/day group males when compared to the control group. Similar increases were not observed in the relative to brain weight ratios, no microscopic lesions that could account for the weight differences were observed and no relationship to treatment was apparent. The mean relative (to final body weight) brain weight in the 500 mg/kg/day group females was significantly lower (p<0.05) than the control group value. This decrease was attributed to the higher final body weight mean in the high dose group and was not considered to be of toxicological significance. The mean relative (to brain weight) heart weight in the 500 mg/kg/day group males was significantly reduced (p<0.05) and the mean relative (to brain weight) liver weight in the 500 mg/kg/day group females was significantly increased (p<0.05) when compared to the control group values. However, there were no microscopic lesions that could account for the weight difference, and the changes were not attributed to treatment. No other remarkable differences in organ weight data were apparent.

GROSS PATHOLOGY:
The 500 mg/kg/day group female that died during study week 2 had gastric ulceration of the non-glandular mucosa and suppurative inflammation in the stomach adjacent to the ulcer. These findings were considered to be a response to a gastric irritant. No other histopathological lesions suggestive of a treatment related effect were observed in the animals that died.

HISTOPATHOLOGY: NON-NEOPLASTIC
At the scheduled necropsy, 1/5 males in the 250 mg/kg/day group and 4/4 females in the 500 mg/kg/day group had gastric submucosal edema. Three of the 500 mg/kg/day group females also had gastric suppurative inflammation. These findings were consistent with a response to a gastric irritant. No other test article related histopathological lesions were observed at any dose level. Hyperkeratosis of the nonglandular gastric mucosa was noted in 212 males in the 500 mg/kg/day group and 1/5 and 1/4 females in the 250 and 500 mg/kg/day groups, respectively. This was considered to be a reflection of the status of food consumption and not necessarily a reflection of a toxic response. Other findings noted in the 250 and 500 mg/kg/day pups were observed at a similar incidence in the control group (such as hypertrophy of the adrenal glands) or were limited to single animals (such as suppurative inflammation in the prostate).

HISTOPATHOLOGY: NEOPLASTIC (if applicable): not applicable.
HISTORICAL CONTROL DATA (if applicable): not applicable.

OTHER FINDINGS
- Organ weights: See above
- Histopathology: See above
- Potential target organs: no data available


Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: rales and salivation (primary or secondary effects due to the local irritation)
Remarks on result:
other: based on local effects (gastric irritation)
Critical effects observed:
not specified

Statistically significant mean body weight losses occurred in the 500 mg/kg/day group males and females during the initial two days of dosing (days 0-2). Body weight gain continued to be inhibited in the high dose group males, but not the females, during study days 2-4 through 10-12 and for the overall study period (days 0-28). No other trends were apparent in body weight change data at a dose level of 500 mg/kg/day. Mean body weights in this group were lower than the control group values for days 2 through 12 in the males and for days 2 and 4 in the females. In the 250 mg/kg/day group, a slightly reduced mean body weight gain was observed in the males during days 0-2. No other adverse effects on body weight data were apparent at dose levels of 250 mg/kg/day and below.

Food consumption (g/animal/day) was slightly reduced, but not statistically significant, in the 500 mg/kg/day group males and females during week 0-1. Food consumption in this group was slightly higher than that in the control group throughout the remainder of the study. No adverse effects on food consumption were apparent in the 10, 50, 125 and 250 mg/kg/day group males and females.

At the scheduled necropsy, thickened mucosa in the nonglandular poltion of the stomach was observed in 1/2 males in the 500 mg/kg/day group and 1/5 and 2/4 females in the 250 and 500 mg/kg/day groups, respectively. At the microscopic examination, 115 males in the 250 mg/kg/day group and 4/4 females in the 500 mg/kg/day group had glandular and/or non-glandular gastric submucosal edema. 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 (to final body weight and to brain weight) adrenal gland weights in the 250 and 500 mg/kg/day group males and females were increased when compared to the control group values. AU of the differences in the males were statistically significant and the differences in the females for relative to brain weight ratios were statistically significant. No test article related histopathological lesions were observed in the adrenal glands to account for the increases.

In conclusion, ZDDP administered orally at a dose level of 500 mg/kg/day produced mortalities (three males and one female), changes in the clinical condition of the animals, inhibition of body weight gain during the first 2-12 days of dosing, inhibition of food consumption during the first week of dosing, increased adrenal gland weights and gross and microscopic gastric tissue changes. In the 250 mg/kg/day group, toxicity was exhibited by changes in the clinical condition of the animals, inhibition of body weight gain in the males during the first two days of dosing, increased adrenal gland weights and gastric tissue changes (gross and microscopic). Several changes in the clinical condition of the animals were observed in the 50 and 125 mg/kg/day group males and females. 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. A dose level of 10 mg/kg/day was considered to be the NOAEL (no observable adverse effect level) for systemic toxicity.

Conclusions:
Repeated oral administration to rats for 28 consecutive days resulted in chemical-related effects at dose levels of 50 to 500 mg/kg/day. A dose level of 125 mg/kg/day was considered to be the NOAEL (no observable adverse effect level) for local effects (gastric irritation).
Executive summary:

The toxicity potential of the test article, Zinc O,O-diethylhexyldithiophosphate (ZDDP), 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 (gastric irritation).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE CATEGORY APPROACH
Please refer also to the read-across statement attached in section 13

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The target and the source substances are structurally similar substances that share the common organometallic core structure consisting of a central zinc metal bonded to four alkyldithiophosphate esters (ligands) by coordinate covalent bonds -Zn[(S2P(OR)2]2. Structural variations between the target and the source substances are related only to the alkyl (R) groups of the alkyldithiophosphate ligands. The substances in this category give thus rise to an (identical) common compound Phosphorodithioic acid moiety that can be released by the breakage of ester bonds and dissociation from the Zinc complex to which the organism would be exposed if the target substance was tested in the toxicity studies. Exposure to the parent compounds (non-transformed constituents) and to the counter alkyl alcohols, possibly released by hydrolysis of P-O bonds – non-common compounds – would not influence the prediction of the (eco)toxicological properties because they are considered to have the same biological targets and to cause the same type of effects through a common underlying mechanism due to the same functional groups (zinc cation, phosphorodithioic cation and aliphatic alcohol anionic moieties). The impurities of the target and the source substances are not expected to impact the prediction because they are identical or, if slightly structural different, belong to the same class of compounds with the same functional groups and their percentages are very low.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
The results of the two source substances indicate that local irritation of the gastrointestinal tract is the primary effect of the substances in the ZDDP category. This is also in line with the skin and eye irritating properties of the substances.
Since the main constituents of the target substance are structurally similar to the constituents of the source substances with the same functional groups and the alkyl chain lengths of phosphoroditioate moieties are in the range of the established ZDDP category (C3-C12), the same mode of toxicological action is expected for the target and the source substances. The constituents of the target substance do not possess functional groups associated with other mode of actions or toxicity effects. Toxicokinetic behavior of the constituents of the target substance is expected to be essentially the same as that of the source substance. Based on the results of the oral repeated dose toxicity studies with the source substances and other ZDDP category members, it is evident that the structural dissimilarities – the chain lengths of the alkyl rests – did not result in different strength of the toxicity effects. Even if absorption through GI tract of the category members with shorter alkyl rests and theoretically higher absorption rates (due to the increased water solubility and decreased molecular weights favouring absorption) is higher, their intrinsic properties are essentially the same as the findings in the toxicity studies are very similar. The impurities of the target substance are considered not to contribute to the toxicity effects because they are also structurally similar to the impurities of the source substances and consist of substances of simple structure without specific mode of action. Furthermore, their amounts are very low. Therefore, it is predicted that the target substance would possess the same toxicity potential by repeated oral exposure as the source substance.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
not specified
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male in the 160 mg/kg/day group was euthanized in extremis on study day 17; a gross observation of a thickened stomach was noted at necropsy. Clinical findings noted for this male approximately 1 hour following dose administration on the day of euthanasia consisted of yellow material on various body surfaces, clear material around the mouth, unkempt appearance, decreased defecation, and labored respiration. Microscopically, this male was noted with inflammation, edema, and ulceration in the non-glandular stomach, erosion and inflammation in the trachea, and lymphoid depletion in the thymus, spleen, and lymph nodes (mesenteric, mandibular, and axillary). The lesions in the non glandular stomach were considered test item-related and may have contributed to the moribund state of this male. All other animals in all dosage groups survived to the scheduled necropsies.
Test item related clinical findings were noted in the 160 mg/kg/day group males and females and included rales, decreased, shallow, and/or labored respiration and salivation related findings. These findings were noted at the daily examinations, at the time of dosing, and/or approximately 1 hour following dose administration primarily during the treatment period. However, because of their sporadic occurrence, these cardio pulmonary findings were considered to be incidental and secondary to the nature of the test item and the route of administration.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights, body weight changes, and food consumption were unaffected by test item administration in the 10, 40, and 160 mg/kg/day groups throughout the treatment and post-treatment periods.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB) AND LOCOMOTOR ACTIVITY (PARENTAL ANIMALS)
No test item-related effects were noted during the FOB or locomotor activity evaluations at any dosage level.

CLINICAL PATHOLOGY (PARENTAL ANIMALS)
There were no test substance-related effects on serum chemistry, hematology, or coagulation parameters in the 10, 40, and 160 mg/kg/day groups.

HAEMATOLOGY
There were no test item-related effects on hematology or coagulation parameters at any dosage level. Significant (p<0.05 or p<0.01) differences were observed when the control and test item-treated groups were compared, and included a higher mean absolute eosinophil count in the 40 mg/kg/day group males at the primary necropsy and a higher mean absolute large unstained cell (LUC) count in the 160 mg/kg/day group females at the recovery (post-treatment) necropsy. These differences were not considered to be test item-related because the values did not show a dose-related response (eosinophils) and were of a magnitude that would be considered to be toxicologically unimportant (LUC). Statistically significant findings that involved percentage reticulocyte or leukocyte differential counts were not itemized above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test item-related alterations in final body weight or organ weights at any dosage level. Significant (p<0.05) differences were observed when the control and high dose group males were compared at the recovery (post-treatment) necropsy and consisted of lower mean kidney weight relative to body weight, higher mean spleen weight relative to brain weight, higher mean left testis weight relative to brain weight, and higher mean right testis weights (absolute and relative to brain weight). There was no case where all 3 measures (absolute, relative to body weight, and relative to brain weight) were statistically significant. Thus, since the absolute weights and weights relative to body or brain weight were discordant, these organ weight changes were considered to be spurious.

GROSS PATHOLOGY (PARENTAL ANIMALS)
One male (no. 61739) in the 160 mg/kg/day group was euthanized in extremis on study day 17. The thickened stomach noted macroscopically for this male was related to test item administration. There were no other test item related internal findings observed for either sex at any dosage level at the scheduled necropsies. Macroscopic findings observed in the test item groups occurred infrequently and/or in a manner that was not dose related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test item-related histologic observations of epithelial hyperplasia, hyperkeratosis, and inflammation of the non-glandular stomach, typically at the limiting ridge, but sometimes more widespread, were noted in the 160 mg/kg/day group males and females.
Dose descriptor:
NOEL
Remarks:
portal-of-entry
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Localised injury to the nonglandular portion of the stomach
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at any dose level.
Critical effects observed:
not specified
Conclusions:
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.
Executive summary:

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subacute
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.

Justification for classification or non-classification

In accordance to Regulation (EC) No 1272/2008 classification of this substance is not required for prolonged exposure.