Zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate)

Endpoint summary

Description of key information

Based on read-across from a structurally similar substance:
Subacute: NOAEL portal-of-entry (rat, m/f) 40 mg/kg bw/day
Subacute: NOAEL systemic toxicity (rat, m/f) 160 mg/kg bw/day (highest dose level)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance CAS 68457-79-4 . According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Neurobehavioral evaluation added to protocol.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Each rat was uniquely identified by a Monel® metal ear tag displaying the animal number.

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 11 weeks old at initiation of treatment; the 10 rats/sex/group used for pairing were approximately 13 weeks old when paired on study day 13.
- Weight at study initiation: 335 g to 429 g (males) and 227 g to 279 g (females); female body weights ranged from 238 g to 319 g on gestation day 0.
- Housing: Following receipt and until pairing for 10 animals/sex/group, all animals were housed individually in clean, stainless steel wire mesh cages suspended above cage board. The cage-board was changed at least 3 times per week. During cohabitation, rats were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding (Bed O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The dams and their litters were housed in these cages until euthanasia on lactation day 4. Females that failed to deliver were housed in plastic maternity cages until post-mating day 25. Males and females not used for pairing remained in suspended stainless steel wire mesh cages until euthanasia.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except during the period of fasting of all males and post-treatment phase females prior to clinical pathology blood collection Feeders were changed and sanitized once per week.
- Water: Reverse osmosis-purified (on site) drinking water, delivered by an automatic watering system ad libitum
- Acclimation period: 13 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior.

ENVIRONMENTAL CONDITIONS
- Temperature: 71.1 to 72.3 deg F (21.7 to 22.4 deg C)
- Humidity: 36.3% to 63.6%
- Air changes: 10 fresh air changes per hour
- Photoperiod: 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod

IN-LIFE DATES: From:8 December 2009 To: 12 February 2010 (Last female necropsy)

Route of administration:

oral: gavage

Vehicle:

other: Mineral Oil USP

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan) once daily.

VEHICLE
- mineral oil, USP
- Supplier: Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ and Gardena, CA locations.
- The vehicle was dispensed into glass container approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature. The vehicle was mixed throughout preparation, sampling, and dose administration procedures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the formulated test item, at concentrations between 0.5 and 20 mg/mL in mineral oil, USP, was established by the Sponsor. Formulations are stable after 10 and 30 days of stationary storage under room temperature conditions. Therefore, stability analyses were not conducted as part of this study.
Quadruplicate samples for homogeneity and concentration analyses were collected from the middle of the vehicle formulation and from the top, middle, and bottom strata of each test item batch formulation. One set of duplicate samples from each batch was shipped under ambient conditions to The Lubrizol Corporation, Wickliffe, OH for dose formulation analyses. The remaining set of duplicate samples was stored under ambient conditions as back-up. The analyzed dosing formulations were within WIL Research’s SOP range for solutions (90% to 110%) and were homogeneous.

Duration of treatment / exposure:

Males: 10/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses.
Females: 10/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. These animals were not evaluated for reproductive parameters.

Frequency of treatment:

Once daily at approximately the same time each day.

Remarks:

Doses / Concentrations:
0, 10, 40, or 160 mg/kg/day administered at a dosage volume of 10 mL/kg.
Basis:
actual ingested

No. of animals per sex per dose:

The low- and mid-dose groups each consisted of 10 rats/sex and the high-dose group consisted of 15 rats/sex. Concurrent control group of 15 rats/sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were determined from the results of previous studies conducted
- Rationale for animal assignment: body weight stratification in a block design using a computer randomization procedure

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Mated females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes, A detailed physical examination was conducted weekly on each animal beginning approximately 3 days prior to the initiation of dose administration. Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for individual animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals. Mated females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB) AND LOCOMOTOR ACTIVITY: Yes, FOB assessments were recorded for 5 animals/sex/group prior to dose administration on study day 27 (males selected for pairing) and on lactation day 4 (females). FOB testing was performed without knowledge of the animal’s group assignment. The FOB was performed in a sound attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB. Home cage, handling, open field, sensory, neuromuscular, and physiological parameters were observed. Forelimb and hindlimb grip strength were measured.
Locomotor activity counts were recorded for 5 animals/sex/group prior to dose administration on study day 27 (males selected for pairing) and on lactation day 4 (females); the same animals evaluated for FOB were selected for locomotor activity assessment. Locomotor activity, recorded after the completion of the FOB, was measured automatically using a personal computer controlled system which utilizes a series of infrared photobeams surrounding a clear plastic, rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the clear plastic boxes to decrease the potential for distraction from extraneous environmental stimuli or stimuli from biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The testing of treatment groups was done according to replicate sequence. Each animal was tested separately. Data were collected in 5 minute epochs and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).

CLINICAL PATHOLOGY: Yes, Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 5 animals/sex/group at the scheduled necropsies (study day 28 for males selected for breeding and lactation day 4 for females) and from 5 animals/sex in the control and high-dose groups following a 14-day non-dosing post-treatment period (study day 42 for males and study day 53 for females). All males (including those not scheduled for clinical pathology assessments) and the post-treatment phase females were fasted overnight prior to blood collection with water available. Blood for serum chemistry and hematology was collected from the retro orbital sinus following isoflurane anesthesia. Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing EDTA (hematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual male body weights were recorded 1 week prior to the initiation of dose administration, on the first day of dose administration, and weekly throughout the study and prior to the scheduled euthanasia. Individual female body weights were recorded beginning approximately 1 week prior to the initiation of dose administration, on the first day of dose administration, and weekly thereafter until evidence of copulation was observed for females selected for pairing. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible), 1, and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Individual food consumption was recorded for both males and females on the corresponding weekly body weight days until pairing. Food consumption continued to be recorded for males and females not selected for pairing until euthanasia. For animals selected for pairing, once evidence of mating was observed, food consumption was recorded for females on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4.

Sacrifice and pathology:

SACRIFICE
- All animals were euthanized by carbon dioxide inhalation. Males selected for pairing were euthanized following completion of the mating period. Males not selected for pairing were euthanized following the 14-day non-dosing post-treatment period. Females that delivered were euthanized on lactation day 4. Females (with evidence of mating) that failed to deliver were euthanized on post mating day 25.

GROSS NECROPSY
- A complete necropsy was conducted on animals euthanized in extremis or at the scheduled necropsies. Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

HISTOPATHOLOGY
- The following tissues were examined microscopically from all treatment phase animals in the control and high dose groups and from the high dose male that was euthanized in extremis. Adrenal glands (2), Aorta, Bone with marrow (sternebrae), Bone marrow smeara, Brain, Cerebrum level 1, Cerebrum level 2, Cerebellum with medul/pons, Coagulating gland, Eyes with optic nerve (2)b, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Liver (sections of 2 lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph node (Axillary, Mesenteric, Mandibular), Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary gland, Prostate gland, Mandibular salivary glands (2), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary glandc, Spinal cord (cervical), Spleen, Testes with epididymidesd (2), Thymus, Thyroids [with parathyroids, if present (2)], Trachea, Urinary bladder, Uteruse with cervix and vagina, All gross lesions (all groups)
In addition, the non-glandular portion of the stomach, thymus, and all gross lesions (all animals) and the testes at all dosage levels were examined microscopically at the scheduled necropsies for both treatment and post treatment phase animals.

ORGAN WEIGHTS
- The following organs were weighed from all F0 animals at the scheduled necropsies: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Spleen, Testes, Thymus gland, Thyroids with parathyroids.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group by sex.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

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:

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.

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: Localized injury to the nonglandular portion of the stomach.

Critical effects observed:

not specified

Executive summary:

In a guideline combined repeated dose and reproduction / developmental screening study (OECD 422) conducted according to Good Laboratory Practices, WIL Research Labs (2010) evaluated the potential toxic effects of phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts 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:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 2) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional group(s) and similarities in physicochemical and/or (eco)toxicological properties (refer to endpoint discussion for further details).
The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6.1, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for grouping of substances and read-across

There are no reliable data available on the repeated dose toxicity of zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate). In order to fulfil the standard information requirements set out in Annex VIII-IX, Section 8.6.1, in accordance with Annex XI, Section 1.5 of Regulation (EC) No 1907/2006, read-across from a structurally similar substance is conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having considered the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, whereby physicochemical, toxicological and ecotoxicological properties may be predicted from data for reference substance(s) by interpolation to other substances on the basis of structural similarity, the substance Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts (CAS 68457-79-4) is selected as reference substances for assessment of repeated dose toxicity.

The read-across is based on structural similarity as both chemicals belong to the substance class of Zinc alkyldithiophosphates (ZDDP) which are produced by a chemical reaction of Phosphorous pentasulfide with low molecular weight alcohols followed by a subseuqent reaction with zinc oxide. A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Overview of repeated dose toxicity:

	Target substance (a)	Source substance (b)
CAS No.	6990-43-8	68457-79-4
Chemical name	zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate)	Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts
Repeated dose toxicity: oral (subacute)	Experimental result: not reliable   RA: CAS 68457-79-4	Experimental result: NOAEL portal-of-entry (rat, m/f) 40 mg/kg bw/day NOAEL systemic toxicity (rat, m/f) 160 mg/kg bw/day (highest dose level; OECD 422)

(a) The substance subject to the REACh Phase-in registration deadline of 31 May 2013 is indicated in bold font. Only for this substance a full set of experimental results and/or read-across is given.

(b) Reference (read-across) substance(s) are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

 

Repeated dose toxicity: oral

Subacute

CAS 6990-43-8

Two subacute (14- and 28-day) studies are available for zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate). However, the composition of the reported test material is unclear and the impurity profile is not specified. It is thus questionable, whether the observed effects were related to the test substance as defined in the substance identity profile or to unidentified impurities in the actual test material.

The 14-day oral toxicity study was conducted in rats and intended as a range finding study for the subsequent 28-day study (Naylor, 1985). Groups of Sprague-Dawley rats (5/sex/dose) were fed a plain diet (control) or diet containing the test material at 100, 500, 1000 and 5000 ppm, 7 days/week for 2 weeks. Based on the reported mean food consumption and body weight data, the achieved dose levels were ca. 10, 53, 103 and 500 mg/kg bw/day (males) and ca. 10, 53, 111 and 493 mg/kg bw/day (females).

There were no or no toxicologically relevant effects on clinical signs, body weight (gain), food intake and gross pathology. The only findings were the following: The brain of one female animal of the 1000 ppm group was hydroencephalic. In the same group, another female animal showed a severe pelvis dilatation (hydronephrosis) on the left side. Mild bilateral hydronephrosis was observed in one female animal of the 5000 ppm group. These effects were deemed to be not treatment-related.

Based on the lack of overall adverse effect, the 14-day oral NOAEL was determined to be 5000 ppm (500 and 493 mg/kg bw/day in male and female rats, respectively).

The 28-day oral toxicity study was performed under GLP conditions and following a protocol similar to OECD guideline 407 (Naylor, 1986). Deviations from the guideline included a limited number of clinical chemistry and pathology parameters determined and no histopathological examinations. The study design included an additional determination of cholinesterase in homogenized brains, plasma and red blood cells. Sprague-Dawley rats (5/sex/dose) were fed plain diet or diet with test material at nominal concentrations of 1000, 2500, 7500 and 10000 ppm (analytical concentrations: 950, 2500, 7400 and 9700 ppm), daily for 4 weeks. Based on analytical concentrations, body weight and food consumption data, the mean dose levels achieved were 83.2, 214.1, 594.7 and 772.2 mg/kg/bw (males) and 93.0, 233.8, 678.5 and 861.9 mg/kg/ bw (females).

Decreased food consumption was observed (primarily in the first week) in animals of the 7500 and 10000 ppm groups. Females at these levels, and to a lesser extent males, showed decreased body weight gain during the study period. There were no adverse clinical observations or gross pathological findings in any test group. Plasma and red blood cell cholinesterase levels were reduced in the 2500, 7500 and 10000 ppm groups. Minor reductions in cholinesterase levels also occurred at the lowest concentration but were not considered to be biologically significant.

The apparent 28-day oral NOEL in male and female rats was thus 1000 ppm, corresponding to 83.2 and 93 mg/kg bw/day in males and females, respectively.

As described above, two subacute (14- and 28-day) studies are available for zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate). However, the composition of the reported test material is unclear and the impurity profile is not specified. There are three 31P-NMR spectra available, the first one (31P-NMR_Rhenocure_349941_161MHz) represents the substance material identity as described in section 1 of this dossier. The spectrum shows the ZDDP typical portions of neutral (peak at 99 ppm) and basic (peaks at 103 and 104 ppm) zinc dithiophosphate.

The second (31P-NMR_Vocol_349942-161MHz) and the third spectrum (31P-NMR Vocol in CDCl3_121MHz) represents the substance identity of the test material which was administered in the 14- and 28 day studies. The degree of impurities is higher in this spectrum, compared to the first spectrum. Non-identified structures are present there, e.g. at 96 ppm ca. 0,7 % (mole% P) in the first spectrum versus 5,9% (Mol% P) in the third spectrum.

All three documents are included as attached background material in the robust study summary of the 28 day study (Naylor, 1986) in section 7.5.1 of Iuclid.

It is thus questionable, whether the observed effects were related to the test substance identity, as defined in the substance identity profile, or to unidentified impurities in the actual test material. Therefore the result of the studies was considered not reliable and unsuitable for assessment.

CAS 68457-79-4

A Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test was conducted with Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts in rats following OECD guideline 422 and under GLP conditions (Coder, 2010). This study is also discussed under Toxicity to reproduction.

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 behaviour, 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.

For the purpose of hazard assessment of zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate) with an average molecular weight of 548.04 g/mol, the lowest reliable dose descriptor available from the source substances is selected as starting point: Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts (CAS 68457-79-4) has an average molecular weight of 576.09 g/mol and a NOAEL oral of 160 mg/kg bw/d (Coder, 2010).

In order to correct the dose descriptor for differences in molar mass, the NOAEL oral of zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate) is estimated to be 152 mg/kg bw/d (= 160 mg/kg bw/d x [548.04/576.09]). This effect level serves as the dose descriptor starting point for derivation of the long-term systemic DNELs for the oral, the dermal and the inhalation route. The DNELs are provided in section 7, Toxicological information.

Conclusions for repeated dose toxicity

Two feeding studies are available in which the repeated dose oral toxicity of zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate) has been investigated.

The 14-day oral NOAEL in male and female rats was 500 and 493 mg/kg bw/day (highest dose level), respectively, based on the lack of overall adverse effects in the parameters examined. Based on decreased food consumption and concomitant reduction in body weight gain, as well as a decrease in plasma and red blood cell cholinesterase levels, the 28-day oral NOEL in male and female rats was 83.2 and 93 mg/kg bw/day, respectively.

Due to unclear composition and impurity profile of the actual test material, the results of these studies are doubtful and considered not reliable and insufficient for assessment.

Therefore, the available data on the structurally similar substance Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts were used for hazard assessment by mean of read-across.

The substance Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts has been tested in a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in rats. Due to test item-related local irritative effects in the nonglandular portion of the stomach at the highest dose level, the local NOAEL (portal-of-entry) was determined to be 40 mg/kg bw/day. No other toxicologically relevant effects were observed up to and including the highest dose level tested and therefore the NOAEL for systemic effects in male and female rats was considered to be 160 mg/kg bw.

Based on read-across, the substance zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate) is considered to be of low toxicity after repeated oral exposure,

There are no data available on repeated dose toxicity by the inhalation and dermal routes.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Justification for classification or non-classification

Based on read-across from a structurally similar substance following an analogue approach, the available data on the repeated dose oral toxicity of the substance do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.