Zinc bis[bis(tetrapropylenephenyl)] bis(hydrogen dithiophosphate)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The substance is classified as a reproductive hazard (Repr. 1B, H360F) based on the presence of dodecylphenol (EC No. 310-154-3) at > 0.3%.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted on structural analogue and suitable for read across. Guideline GLP study.

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)

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 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.

Details on mating procedure:

The 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0.

For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

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 sponsor 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
gavage

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

Parental animals: Observations and examinations:

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 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 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 (if feeding study): 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 paiting, 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.

Litter observations:

PARTURITION
All females were allowed to deliver naturally and rear their young to PND 4. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

LITTER VIABILITY AND DEATHS
Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring dying were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. The carcass of each pup was then discarded.

CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nesting and nursing behavior were recorded.

BODY WEIGHTS
Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data.

SEX DETERMINATION
Pups were individually sexed on PND 0 and 4.

Postmortem examinations (parental animals):

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.

Postmortem examinations (offspring):

SACRIFICE
On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded without examination.

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.

Reproductive indices:

Reproductive indices were calculated for: Male Mating Index (%), Female Mating Index (%), Male Fertility Index (%), Female Fertility Index (%), Male Copulation Index (%), Female Conception Index (%), Pre-Coital Interval (days).

Offspring viability indices:

Litter parameters were calculated for: Mean Live Litter Size, Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter), Postnatal Survival for All
Other Intervals (% Per Litter).

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels.

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:

clinical signs

Remarks on result:

other:

Remarks:

Localized 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:

clinical signs

Remarks on result:

other:

Remarks:

No effects observed at any dose level

Dose descriptor:

NOEL

Remarks:

reproductive

Effect level:

160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other:

Remarks:

No effects observed at any dose level.

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
Mean numbers of corpora lutea and unaccounted-for sites, mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 10, 40, and 160 mg/kg/day groups were similar to the control group values.

CLINICAL SIGNS (OFFSPRING)
The general physical condition of all F1 pups in this study were unaffected by test item administration. No test item-related clinical findings were noted for the F1 pups.

BODY WEIGHT (OFFSPRING)
Mean male and female pup body weights and body weight gains in the 10, 40, and 160 mg/kg/day groups were unaffected by test item administration during PND 1-4. No statistically significant differences from the control group were noted.

GROSS PATHOLOGY (OFFSPRING)
There were no remarkable macroscopic findings in the F1 pups at the scheduled necropsy at any dosage level.

Dose descriptor:

NOEL

Remarks:

neonatal toxicity

Generation:

F1

Effect level:

160 mg/kg bw/day (actual dose received)

Based on:

other: maternal dose

Sex:

male/female

Basis for effect level:

viability

Remarks on result:

other:

Remarks:

no effects observed at any dose level.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

EC 234-277-6 has not been tested for reproduction toxicity, however experimental data on structurally related substances EC 270-608-0 was available and suitable for read-across.
Based on this study, NOAEL of 160 mg/kg/day for reproduction toxicity was proposed for EC 234-277-6

Executive summary:

In a guideline 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.

Under the conditions of this screening study, no test material-related effects on reproductive performance, gestation length, parturition, reproductive organs, or neurobehavioral parameters were noted at any dosage level. Based on these results, a dosage level of 160 mg/kg/day was considered to be the no-observed-effect level (NOEL) for reproductive toxicity. 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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

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:

clinical signs

Remarks on result:

other:

Remarks:

localised injury to the nonglandualr 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:

clinical signs

Remarks on result:

other:

Remarks:

no effects observed at any dose level

Dose descriptor:

NOEL

Remarks:

reproductive

Effect level:

160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other:

Remarks:

no effects observed at any dose level

Critical effects observed:

no

Dose descriptor:

NOEL

Remarks:

neonatal toxicity

Generation:

F1

Effect level:

160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

viability

Remarks on result:

other:

Remarks:

no effects observed at any dose level

Critical effects observed:

no

Reproductive effects observed:

no

Additional information

Where tetrapropenyl phenol (a.k.a. dodecylphenol, EC No. 310-154-3) is expected to be present in excess of 0.3 %, this UVCB substance is classified as a reproductive hazard (Repr 1B, H360F). Dodecylphenol has been found to affect fertility in 1 and 2-generation reproductive toxicity studies but did not adversely impact development.

The zinc dialkyldithiophosphate present in phenol, tetrapropenyl-, hydrogen phosphorodithioate, zinc is not expected to cause reproductive or developmental toxicity based on read across to structurally related category substances EC 224-235-5 and EC 270-608-0.

 The reproductive toxicity of an analogue of this substance, EC 224-235-5, was evaluated with rats at doses as high as 200 mg/kg/day in accordance with OECD Guideline 421. Adverse effects on reproduction were observed only at doses that caused maternal toxicity. Treatment-related mortality and clinical signs were noted in the parents at dose levels of 100 and 200 mg/kg/day by mortality and clinical signs. Treatments-related inhibition of body weight gain in males and signs of gastric irritation also was observed at the 200 mg/kg/day dose level. No parental toxicity was found at the 30 mg/kg/day dose level. Slightly reduced fertility indices were observed at the 200 mg/kg/day dose level. Reproductive performance (fertility, mating, days between pairing and coitus, gestation and parturition) was unaffected by treatment at the 30 and 100 mg/kg/day dose levels. Based on the results of this study, a dose level of 30 mg/kg/day was considered to be the NOAEL (no observable adverse effect level) for parental and neonatal toxicity.

The reproductive toxicity of a second analogue of this substance, EC 270-608-0, was evaluated with rats at doses as high as 160 mg/kg/day for up to 52 consecutive days in accordance with OECD Guideline 422. No substance-related effects on reproductive performance, gestation length, parturition, reproductive organs, or neurobehavioral parameters were found. Substance-related moribundity, clinical findings, and epithelial hyperplasia, hyperkeratosis, and inflammation of the glandular portion of the stomach were observed in the 160 mg/kg/day group. The injury to the stomach was localized and was considered to be a portal of entry effect due to the irritation of the test material. The NOEL for portal-of-entry effects was determined to be 40 mg/kg/day. The NOEL for reproductive toxicity was determined to be 160 mg/kg/day. The parental NOAEL for systemic toxicity also was determined to be 160 mg/kg/day.

The potential for residual starting material (dodecylphenol, EC No. 310-154-3) to be present in the registered substance has been taken into account for risk assessment purposes. Where dodecylphenol, EC No. 310-154-3, is present at significant concentrations, it has been assumed that this will result in a classification of Repr. IB, H360F, in accordance with the harmonised Annex VI entry for this substance. However, for the purpose of a robust risk assessment in accordance with the conditions of Point 8.7.3 of Column 2 in Annex IX of Regulation EC 1907/2006, the information that will be generated as indicated in this testing proposal is considered to be relevant to the risk assessment for this material. The zinc dialkyldithiophosphate represents the majority of the composition, and as such is relevant for inclusion.

Effects on developmental toxicity

Description of key information

The substance is classified as a reproductive hazard (Repr. 1B, H360F) based on the presence of dodecylphenol (EC No. 310-154-3) at > 0.3%.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted on structural analogue and suitable for read across. Guideline GLP study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

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 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 sponsor 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.

Details on mating procedure:

The 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0.

For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

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.

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

Maternal examinations:

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 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 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 (if feeding study): 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 paiting, 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
- 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.

Indices:

Litter parameters were calculated for: Mean Live Litter Size, Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter), Postnatal Survival for All Other Intervals (% Per Litter).

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: portal-of-entry

Details on maternal toxic effects:
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.

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.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other:

Remarks:

localized 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.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other:

Remarks:

no effects observed at any dose level.

Abnormalities:

no effects observed

Dose descriptor:

NOEL

Effect level:

160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

other: development toxicity

Remarks on result:

other:

Remarks:

no effects on general physical condition of F1 pups

Abnormalities:

no effects observed

Developmental effects observed:

no

Results for EC 270 -608 -0

VIABILITY (OFFSPRING): Mean numbers of corpora lutea and unaccounted-for sites, mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 10, 40, and 160 mg/kg/day groups were similar to the control group values.

CLINICAL SIGNS (OFFSPRING): The general physical condition of all F1 pups in this study were unaffected by test item administration. No test item-related clinical findings were noted for the F1 pups.

BODY WEIGHT (OFFSPRING): Mean male and female pup body weights and body weight gains in the 10, 40, and 160 mg/kg/day groups were unaffected by test item administration during PND 1-4. No statistically significant differences from the control group were noted.

GROSS PATHOLOGY (OFFSPRING): There were no remarkable macroscopic findings in the F1 pups at the scheduled necropsy at any dosage level.

JUSTIFICATIONS FOR READ-ACROSS

EC 272-723-1 has not been tested for development toxicity, however experimental data on structurally related substances EC 270-608-0 was available and suitable for read-across. Justifications for read-across:

Manufacture/Usage:

EC 272-723-1 and EC 270-608-0 are generically referred to as zinc dialkylthiophosphate (ZDDP) which are produced under similar manufacturing procedures and used in commerce as multi-functional anti-wear and anti-oxidation inhibitor performance components in passenger motor oils, diesel engine oils and industrial oils such as hydraulic lubricants.

Chemical Similarity:

EC 272-723-1 and EC 270-608-0 consist of alkyl substituted phosphorodithioic acid structures complexed with zinc.

EC 272-723-1:Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-propyl) esters, zinc salts, referred to as “mixed 2-ethylhexyl and isopropyl derivative”

EC 270-608-0:Phosphorodithioic acid, mixed O,O-bis(iso-butyl and pentyl) esters, zinc salts, referred to as “mixed isobutyl and pentyl derivative”

These ZDDPs share similar core structures - alcohol ester of dithiophosphate, specific structural variations that relate to their alcohol group substituent are the alkyl chain length and the degree of branching of the alcohol charge. Using Tanimoto Fingerprint (ToxMatch Version 1.06 software) to model chemical structures of the analogues showed comparable values for relevant molecular descriptors (e.g., number of H bond acceptor atoms), and gave a similarity index greater than 0.8 (values range from 0, no similarity to 1, identical; peer reviewed literature indicates that values greater than 0.6 are significantly similar); therefore chemical structures of the analogues have determined to be sufficiently close for there to be a reasonable expectation of similar toxicological effects.

Physicochemical Properties:

Standard physicochemical properties for each substance were listed in Table 1. 

Table 1. Establishment of similarity between the data donating substance and the data accepting substance

EC	Alkyl Group	Average MW	log Kow	Water Sol (ppm)	Vapor Pressure (Pa at 25 oC)
272-723-1  (accept data)	C3/C8 branched	632	0.84	2111	1.9 x10^-3
270-608-0 (donate data)	C4, branched C5, mixture of linear and branched	576	0.69	1658	2.5 x10^-3

As shown in Table 1, these two substances have similar molecular weight and Log Kow, low vapor pressure, and both are water soluble. The similarity of the physicochemical properties of these substances parallels their structural similarity.

Biologically Active Functional Groups:

The ester group presents in each of the analogue members, and is expected to exhibit similar biological activities. Non-random patterns were observed for the toxicological effects(e.g. available data showed low levels of acute toxicity effect, lacking of mutagenic effect in bacteria, lacking of cryptogenic effects in rats, consistent trend of change in ecotoxicity effect, etc.), these common behaviors and consistent trends suggest a common mechanism/mode of action, with little influence from the length of carbon chain. These facts further supported read-across between the analogue members.

Available Data and Adequacy for Read-across:

The developmental toxicity of EC 270-608-0 was evaluated with rats at doses as high as 160 mg/kg/day for up to 52 consecutive days in accordance with OECD 422. No substance-related effects on reproductive performance, gestation length, parturition, reproductive organs, or neurobehavioral parameters were found. Substance-related toxicity was limited to morbundity, adverse clinical signs, and epithelial hyperplasia, hyperkeratosis, and inflammation of the stomach. The NOAEL and NOEL for reproductive fertility and neonatal toxicity was determined to be 160 mg/kg/day. The parental NOAEL for systemic toxicity was 160 mg/kg/day. The parental NOAEL for portal of entry irritation and related secondary effects parental toxicity was 40 mg/kg/day.

Conclusion:

Based on the abovementioned justifications, results from EC 270-608-0 OECD 422 study was determined to be adequateto fulfill the purposes of this endpoint.Therefore, NOAEL of 160 mg/kg/day for reproductive/developmental toxicity was proposed for EC 272-723-1, and use of read-across will eliminate the need for new animal testing while allow meeting the data requirements.

 

Conclusions:

EC 234-277-6 has not been tested for developmental toxicity, however experimental data on structurally related substances EC 270-608-0 was available and suitable for read-across.
Based on this study, NOAEL of 160 mg/kg/day for reproduction/development toxicity was proposed for EC 234-277-6.

Executive summary:

In a guideline 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 saltswhen 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. In the absence of effects on the general physical condition of the F1pups, the NOEL for neonatal toxicity was 160 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOEL

Remarks:

portal-of-entry

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other:

Remarks:

localised injury to the nonglandular portion of the stomach

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other:

Remarks:

no effects observed at any dose level

Abnormalities:

no effects observed

Dose descriptor:

NOEL

Effect level:

160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other:

Remarks:

no effects on general physical condition of F1 pups

Abnormalities:

no effects observed

Developmental effects observed:

no

Additional information

The potential for residual starting material (dodecylphenol, EC No.310-154-3) to be present in the registered substance has been taken into account for risk assessment purposes. The RAC decision concerning dodecylphenol, EC No. 310-154-3, for its inclusion on Annex VI (9th ATP) considered long-term systemic effects including developmental toxicity.  From this evaluation, it was concluded that the material does not meet the criteria for classification for developmental effects and in this case is not relevant for classification and labelling. As such it is considered appropriate that the risk assessment for this endpoint is based on the zinc dialkyldithiophosphate which represents the majority of the composition of this substance.

The potential of an analogue of this substance, EC 224-235-5, to affect development was evaluated with rats at doses as high as 200 mg/kg/day in accordance with OECD Guideline 421. Adverse effects on development were observed only at doses that caused maternal toxicity. Treatment-related mortality and clinical signs were noted in the parents at dose levels of 100 and 200 mg/kg/day by mortality and clinical signs. Treatments-related inhibition of body weight gain in males and signs of gastric irritation also was observed at the 200 mg/kg/day dose level. No parental toxicity was found at the 30 mg/kg/day dose level. Neonatal mortality in the F1 generation was observed at the 100 and 200 mg/kg/day dose levels. Neonatal clinical signs of toxicity also were noted at the 200 mg/kg/day dose level as evidenced by clinical signs. No neonatal toxicity was observed at a dose level of 30 mg/kg/day. Based on the results of this study, a dose level of 30 mg/kg/day was considered to be the NOAEL (no observable adverse effect level) for parental and neonatal toxicity.

The developmental toxicity of an analogue of this substance, EC 270-608-0, was evaluated with rats at doses as high as 160 mg/kg/day for up to 52 consecutive days in accordance with OECD Guideline 422. No substance-related effects on postnatal survival, bogy weights, body weight gain, or general physical appearance of the pups were found. Substance-related morbundity, clinical findings, and epithelial hyperplasia, hyperkeratosis, and inflammation of the glandular portion of the stomach were observed in the 160 mg/kg/day group. The injury to the stomach was localized and was considered to be a portal of entry effect due to the irritation of the test material. The NOEL for portal-of entry effects was determined to be 40 mg/kg/day. The NOEL for neonatal toxicity was determined to be 160 mg/kg/day. The parental NOAEL for systemic toxicity also was determined to be 160 mg/kg/day.

Justification for classification or non-classification

In accordance with EU CLP (Regulation (EC) No. 1272/2008), classification of this substance is required for reproductive toxicity, Repr 1B. (H360F) when dodecylphenol (EC No. 310 -154 -3) is present at > 0.3%. When the level of dodecylphenol is < 0.3%, classification is not required, based on read-across to the available data on analogue substances.