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

Endpoint summary

Description of key information

Repeat dose oral toxicity data is not available for EC 234-277-6, but an OECD 422 (NOAEL 160 mg/kg/d) and an OECD 407 (NOAEL 125 mg/kg/d) study are available for analogous substances EC 270-608-0 and EC 224-235-5, respectively. These studies were suitable for read across, and the lower NOAEL, 125 mg/kg/d, was used as the NOAEL for EC 234-277-6.  

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

29 October 1993 - 30 August 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study compliant with standardised tests, and is sufficiently detailed. Study was conducted on structural analogue and suitable for read across.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

A functional observational battery for neurotoxicity was not performed ; this test not part of the OECD 407 guideline at time of study. Hematology and clinical chemistry parameters were not evaluated. Limited microscopic pathology performed.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Source: Charles River Breeding laboratories, Inc, Portage, Michigan.
-Age at study initiation: 10 weeks old (male) and 11 weeks old (female)
-Weight at study initiation: At the start of treatment the males weighed ranged from 327 to 415 g, and the females weighed ranged from 237 to 272 g.
-Fasting period before study: none.
-Housing: Upon arrival, all animals were housed three per cage by sex for a minimum of three days. Thereafter, all animals were housed individually in clean, wire-mesh cages suspended above cage-board.
-Diet (e.g. ad libitum): The animals were allowed free access to food. The basal ration used in this study was Purina" Certified Rodent Chow® #5002. The diet utilized at WIL Research Laboratories, Inc., is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research Laboratories, Inc.
-Water (e.g. ad libitum): free access to tap-water. Municipal water supplying the facility is sampled and analyzed for contaminants according to Standard Operating Procedures.

-Acclimation period: 15 days.

ENVIRONMENTAL CONDITIONS
-Temperature (°C): 18 to 22 oC
-Humidity (%): 22% to 78%
-Air changes: no data available.
-Photoperiod: twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: October 28, 1993 To: December 10, 1993

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: A sufficient amount of Mazoia® Corn Oil to dose the control group animals was placed in a properly labeled storage container. The vehicle control article was stirred continuously throughout the sampling and dosing procedures using a magnetic stir bar and plate.

The appropriate amount of test article, ZDDP, for each group was weighed into tared, precalibrated, storage containers. Mazoia® Corn Oil was added to the calibration mark. The test article preparations were stirred continuously throughout the sampling and dosing procedures using magnetic stir bars and plates. The dose formulations were visually inspected for homogeneity prior to dosing.
All dosing preparations were made weekly and were stored at room temperature between periods of use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance formed a homogenous solution in corn oil, and this preparation was stable for at least 7 days.
- Concentration in vehicle: 0, 2, 10, 25, 50, 100 mg/ml.
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): NDA
- Purity: 100%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All analyses were performed by Chevron Research and Technology Company, Richmond, California. Prior to study initiation, two sets of samples from the middle of the control group and four sets of samples each from the top, middle and bottom of a 2 mg/ml and a 100 mg/ml formulation prepared prior to dose level selection were collected. One-half of the samples were analyzed for homogeneity. The remaining samples were stored under normal laboratory conditions for eight days then analyzed to verify stability of the test article in the vehicle. From each weekly formulation, two sets of samples from the middle of the control group and four sets of samples from the middle of the 10, 50, 125, 250 and 500 mg/kg/day groups were collected. One-half of the samples were analyzed for concentration. The results of these analyses are the responsibility of the sponsor and will be reported separately by the sponsor. The remaining samples were stored at WIL Research Laboratories, Inc., until conformation of receipt of intact samples by the sponsor.

Duration of treatment / exposure:

The test material was administered daily, for twenty-eight consecutive days.

Frequency of treatment:

Once daily, 7 days per week.

Remarks:

Doses / Concentrations:
10 mg/kg/day active ingredient
Basis:
actual ingested

Remarks:

Doses / Concentrations:
50 mg/kg/day/active ingredient
Basis:
actual ingested

Remarks:

Doses / Concentrations:
125 mg/kg/day active ingredient
Basis:
actual ingested

Remarks:

Doses / Concentrations:
250 mg/kg/day active ingredient
Basis:
actual ingested

Remarks:

Doses / Concentrations:
500 mg/kg/day active ingredient
Basis:
actual ingested

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels based upon a preliminary range finding study. The 5-day range finding study was performed with 3 rats/sex/group at oral dose levels of 1000 mg/kg/day to provide a basis for selection of dose levels for the 28-day study.
The test material was prepared as a solution in corn oil, and was administered orally as a single daily dose for five consecutive days. All animals died or were euthanized in extremis prior to study day 5. Based on the results of this study, dose levels of 10, 50, 125, 250 and 500 mg/kg/day were selected for the dose range-finding toxicity study of ZDDP in rats.

- Rationale for animal assignment (if not random): all available rats were weighed and examined in detail for physical abnormalities. Animals judged suitable for testing, as decided by the study director, were selected for use for the computerized randomization procedure. The individual body weights and corresponding animal numbers were entered into the WE Toxicology Data Management System (WTDMSTD). A printout containing the animal numbers, corresponding body weights and individual group assignments was generated based on body weight stratif~cation in a block design. The animals then were arranged according to the printout. The control and five test groups each consisted of five males and five females.

- Rationale for selecting satellite groups: no data available.
- Post-exposure recovery period in satellite groups: no data available.
- Section schedule rationale (if not random): Random

Positive control:

not used

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
- Cage side observations checked in table [No.?] were included. No.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed on all animals prior to dosing, at the time of dosing and approximately 1 hour following dosing with the following exceptions. AU animals were observed for clinical signs one and two hours following dosing on November 13 and 14, 1993 (study days 1 and 2), and all animals were observed approximately two hours following dosing, rather than approximately one hour following dosing, on November 23, 1993 (study day 11). All observations were recorded.
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on study days -7, 0 and every other day after until study termination (day 28). Mean body weight changes were calculated for each interval and also for study days 0-8, 8-14, 14-22, 22-28 and 0-28. A final body weight was recorded for each animal on the day of scheduled necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, individual food consumption was measured weekly beginning one week prior to test article administration (week -1). Food intake was calculated as g/animal/day.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data.
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: no data available.
- Dose groups that were examined: no data available.

HAEMATOLOGY: No
- Time schedule for collection of blood: no data available.
- Anaesthetic used for blood collection: no data available.
- Animals fasted: no data available.
- How many animals: no data available.
- Parameters were examined: no data available.

CLINICAL CHEMISTRY: No
- Time schedule for collection of blood: no data available.
- Animals fasted: no data available.
- How many animals: no data available.
- Parameters were examined: no data available.

URINALYSIS: No
- Time schedule for collection of urine: no data available.
- Metabolism cages used for collection of urine: no data available.
- Animals fasted: no data available.
- Parameters were examined: no data available.

NEUROBEHAVIOURAL EXAMINATION: No.
- Time schedule for examinations: no data available.
- Dose groups that were examined: no data available.
- Battery of functions tested: no data available.

OTHER: overt signs of toxicity, ill health or behavioural change were examined immediately before dosing and one hour after dosing..

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete necropsy was conducted on all animals that died or were anesthetized at study termination. All animals were anesthetized by carbon dioxide asphyxiation and exsanguinated. The necropsy included, but was not limited to, examination of the extend surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and fixed:
Adrenals, Aorta (thoracic), Bone with marrow (stenebrae), Bone marrow smear (from femur), Brain (forebrain, midbrain, hindbrain), Eyes with optic nerve (2) Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, HeaIt, Kidneys (2), Liver (sections of two lobes), Lungs [including bronchi, futed by inflation with fixative (2)], Lymph node (mesenteric), Mammary gland (females only) , Ovaries with oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary, Prostate, Salivary glands [submaxillary (2)], Seminal vesicles (2), Skeletal muscle (vastus medialis), Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Testes with epididymides (2), Thymus, Thyroid [with parathyroids if present (2)], Trachea, Urinary, bladder, Uterus with vagina, and all gross lesions.

Other examinations:

ORGAN WEIGHTS
The following organs from animals euthanized at study termination were weighed:
Adrenals,Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Testes.
Paired organs were weighed collectively. Organ to final body weight ratios and organ to brain weight ratios were calculated.

MICROSCOPIC EXAMINATION
After fixation, specified tissues were trimmed as described by Thompson2. Trimmed specimens were placed in appropriately labeled and numbered cassettes. The tissue samples were processed and then embedded in paraffin blocks. The labeled paraffin blocks were sectioned at five to eight microns. The tissue sections were placed on clean glass microscopic slides labeled with the appropriate study, animal, group and cassette number. Upon completion of the staining with hematoxylin and eosin (AFIP Manual of Histological Staining Methods3), cover slips were placed on the slides.
The esophagus, stomach, intestine (small and large), gonads, accessory sex tissues and gross Lesions were examined microscopically from all animals that died and from the animals in the control and 500 mg/kg/day groups euthanized at the scheduled necropsy. Due to the mortality observed in the 500 mg/kg/day group, the previously mentioned tissues were also examined microscopically from the 250 mg/kg/day group animals at the scheduled necropsy.

The adrenal glands were examined microscopically from animals in the control, 250 and 500 mg/kg/day groups due to an increase in absolute adrenal gland weights at these dose levels.

Statistics:

All analyses were conducted using two tailed tests for minimum significance levels of 1% and 5 % comparing the treatment groups to the vehicle control group by sex. AU means were presented with standard deviations (S.D.) and the numbers of sampling units (N) used to calculate the means. All statistical tests were performed by a Digital® MicroVAX® 3400 computer with appropriate programming. Analysis of body weight, body weight change, food consumption, and absolute and relative organ weight data were subjected to a one-way analysis of variance, followed by Dunnett's test.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Three males in the 500 mg/kg/day group died, one 500 mg/kg/day group female died. The most remarkable clinical signs observed prior to death in these animals consisted of hypoactivity, body cool to touch, hunched appearance, unkempt appearance, extremities pale in color and/or respiratory distress (rales, labored respiration, shallow respiration). One female in the 125 mg/kg/day group died. This female was noted to have the oral cavity obstructed by a thick brown material on the day of death. Other remarkable clinical signs for this female prior to death included hypoactivity, body cool to the touch, hunched appearance, unkempt appearance, extremities pale in color, excessive chewing and respiratory distress (rales, labored respiration, shallow respiration).
All other animals survived to the scheduled necropsy. The predominant clinical signs, observed at a similar incidence and severity throughout the study, consisted of the following. Changes in fecal consistency (soft stool, diarrhea, mucoid diarrhea, mucoid feces), discoloration of the feces and tan, yellow or brown staining on various body surfaces were observed in the 125,250 and 500 mg/kg/day group males and females, primarily at the daily examinations prior to dosing. Rales occurred in the 50 mg/kg/day group males and the 125, 250 and 500 mg/kg/day group males and females, primarily at the daily examinations and one hour following dosing. Salivation was observed in these same dose groups at the time of dosing. Aggression was noted in the 125, 250 and 500 mg/kg/day group males and females at the time of dosing.

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

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

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

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

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

FOOD EFFICIENCY
No data.

WATER CONSUMPTION
No data.

OPHTHALMOSCOPIC EXAMINATION:
No data.

HAEMATOLOGY
No data.

CLINICAL CHEMISTRY
No data.

URINALYSIS
No data.

NEUROBEHAVIOUR
No data.

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

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

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

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

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

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

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

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

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

In conclusion, ZDDP administered orally at a dose level of 500 mg/kg/day produced mortalities (three males and one female), changes in the clinical condition of the animals, inhibition of body weight gain during the first 2-12 days of dosing, inhibition of food consumption during the first week of dosing, increased adrenal gland weights and gross and microscopic gastric tissue changes. In the 250 mg/kg/day group, toxicity was exhibited by changes in the clinical condition of the animals, inhibition of body weight gain in the males during the first two days of dosing, increased adrenal gland weights and gastric tissue changes (gross and microscopic). Several changes in the clinical condition of the animals were observed in the 50 and 125 mg/kg/day group males and females. Based on the data obtained, a dose level of 500 mg/kg/day would exceed a maximum tolerated dose for a subsequent reproduction/developmental toxicity study in rats.Based on these findings, the systemic and local portal-of-entry no observed adverse effect level (NOAEL) was determined to be 125 mg/kg/day.

 

 

Read-Across Justification for EC 234-277-6

EC 234-277-6 has not been tested for repeat dose toxicity, however study with the structurally related substance EC 224-235-5 was available and suitable for read-across.

Consistent with ECHA and OECD Guidance, read-across can be performed to fill data gaps for a substance when one or more analogues have similarity from multiple lines of evidence including structural, physical-chemical, mechanistic, toxicological and/or ecotoxicological bases.^1-3

The registered substance EC 234-277-6 is a member of the group of inter-related ZDDP substances of similar structure and chemical properties that have previously been assessed as a category under the HPV program. For the purposes of read-across to fill data gaps for this substance the analogue EC 224-235-5, zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate), is justified for use based on its similar structure, physical chemical properties, and fate and effects profile. For some endpoints where multiple reliable analogs exist, “worst case” data is selected based on the most precautionary test result, or based on reading across from lower molecular weight to higher or from higher water solubility to lower.

The following discussion provides multiple lines of evidence justifying this read across approach:

I. Category: EC 234-277-6 substance and EC 224-235-5 analog have been demonstrated to show sufficient structural and physicochemical similarity to be included in the High Production Volume (HPV) Chemical Challenge Program under the Zinc Dialkyldithiophosphate (ZDDP) category.

II. Manufacture/Usage:  EC 234-277-6 substance and EC 224-235-5 analog are substances that are generically referred to as zinc dialkylthiophosphate (ZDDP) that are produced under similar manufacturing procedures and are intended for multifunctional use as oil additives for antioxidancy and antiwear.

III. Chemical Similarity:EC 234-277-6 substance and EC 224-235-5 analog have the general empirical formula of C#H#O4P2S4Zn and are coordination complexes of zinc metal bonded to alkyldithiophosphate ligands. ZDDP complexes exist in reversible monomeric or dimeric forms (equilibrium dependent on temperature) and a basic form. The stereochemistry of the basic form can be described as four Zn atoms arranged around a tetrahedral oxide with six alkyldithiophosphate ligands. As a group, these ZDDPs share similar alcohol ester of dithiophosphate core structures, and variations that relate to alkyl chain length and the degree of branching of the alcohol. Using Tanimoto Fingerprint (ToxMatch Version 1.06 software) to model the chemical structures of the substances and its analog 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 and read-across is supported.

IV. Physicochemical Properties:EC 234-277-6 substance and EC 224-235-5 analog have similar values for average molecular weight (based on the monomer structure), log Kow, water solubility, and vapor pressure; or in some instances for read-across purposes “worst case” values are selected by going from a lower to a higher molecular weight, or from a higher to a lower water solubility.

V. Biologically Active Functional Groups: The ester group is a common functional group present in each of the analogue members, and is expected to exhibit similar biological activities with little influence from the length of carbon chain. Any potential breakdown products, via physical or biological processes, are also expected to result in structurally similar chemicals. In addition, non-random patterns have been observed for the toxicological effects (e.g., available data showed low levels of acute toxicity, lack of mutagenic potential, and a trend of change in ecotoxicity potential based on molecular weight). These common behaviors and consistent trends suggest a common mechanism and mode of action thereby providing further supporting evidence for the read-across among the ZDDP members.

 

 

REFERENCES:

1. ECHA Chapter R.7a: Endpoint specific guidance. Guidance on Information Requirements and Chemical Safety Assessment, http://wko.at/up/enet/chemie/TL_ChapterR7a.pdf;

2. ECHA Practical Guide 6: How to Report Read-Across and Categories,http://echa.europa.eu/doc/publications/practical_guides/pg_report_readacross_categ.pdf;

3. OECD 2007. Guidance on grouping of chemicals. ENV/JM/MONO(2007)28

 

Conclusions:

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

Executive summary:

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

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

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

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

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

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

other:

Remarks:

Dosing animal with 125, 250 and 500 mg/kg/d, reduction in body weight gain, histopatholigical lesions in stomach, rales and salivation (also seen in 50 mg/kg/d test group), etc. were observed. These findings are primary or secondary effects due to the local irritation via the experimental route of administration, so NOAEL is determined to be 125 mg/kg/d.

Critical effects observed:

no

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting 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)

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 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

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 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 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:

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.

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at any dose level.

Critical effects observed:

not specified

Read-Across Justification for EC 234-277-6

EC 234-277-6 has not been tested for repeat dose toxicity, however experimental data from a structurally related substance EC 270-608-0 was available and suitable for read-across.

Consistent with ECHA and OECD Guidance, read-across can be performed to fill data gaps for a substance when one or more analogues have similarity from multiple lines of evidence including structural, physical-chemical, mechanistic, toxicological and/or ecotoxicological bases.^1-3

The registered substance EC 234-277-6 is a member of the group of inter-related ZDDP substances of similar structure and chemical properties that have previously been assessed as a category under the HPV program. For the purposes of read-across to fill data gaps for this substance the analogue ZDDP EC 270-608-0, phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts, is justified for use based on its similar structure, physical chemical properties, and fate and effects profile. For some endpoints where multiple reliable analogs exist, “worst case” data is selected based on the most precautionary test result, or based on reading across from lower molecular weight to higher or from higher water solubility to lower.

The following discussion provides multiple lines of evidence justifying this read across approach:

I. Category: EC 234-277-6 substance and EC 270-608-0 analog have been demonstrated to show sufficient structural and physicochemical similarity to be included in the High Production Volume (HPV) Chemical Challenge Program under the Zinc Dialkyldithiophosphate (ZDDP) category.

II. Manufacture/Usage:  EC 234-277-6 substance and EC 270-608-0 analog are substances that are generically referred to as zinc dialkylthiophosphate (ZDDP) that are produced under similar manufacturing procedures and are intended for multifunctional use as oil additives for antioxidancy and antiwear.

III. Chemical Similarity:EC 234-277-6 substance and EC 270-608-0 analog have the general empirical formula of C#H#O4P2S4Zn and are coordination complexes of zinc metal bonded to alkyldithiophosphate ligands. ZDDP complexes exist in reversible monomeric or dimeric forms (equilibrium dependent on temperature) and a basic form. The stereochemistry of the basic form can be described as four Zn atoms arranged around a tetrahedral oxide with six alkyldithiophosphate ligands. As a group, these ZDDPs share similar alcohol ester of dithiophosphate core structures, and variations that relate to alkyl chain length and the degree of branching of the alcohol. Using Tanimoto Fingerprint (ToxMatch Version 1.06 software) to model the chemical structures of the substances and its analog 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 and read-across is supported.

IV. Physicochemical Properties:EC 234-277-6 substance and EC 270-608-0 analog have similar values for average molecular weight (based on the monomer structure), log Kow, water solubility, and vapor pressure; or in some instances for read-across purposes “worst case” values are selected by going from a lower to a higher molecular weight, or from a higher to a lower water solubility.

V. Biologically Active Functional Groups: The ester group is a common functional group present in each of the analogue members, and is expected to exhibit similar biological activities with little influence from the length of carbon chain. Any potential breakdown products, via physical or biological processes, are also expected to result in structurally similar chemicals. In addition, non-random patterns have been observed for the toxicological effects (e.g., available data showed low levels of acute toxicity, lack of mutagenic potential, and a trend of change in ecotoxicity potential based on molecular weight). These common behaviors and consistent trends suggest a common mechanism and mode of action thereby providing further supporting evidence for the read-across among the ZDDP members.

 

REFERENCES:

1. ECHA Chapter R.7a: Endpoint specific guidance. Guidance on Information Requirements and Chemical Safety Assessment, http://wko.at/up/enet/chemie/TL_ChapterR7a.pdf;

2. ECHA Practical Guide 6: How to Report Read-Across and Categories,http://echa.europa.eu/doc/publications/practical_guides/pg_report_readacross_categ.pdf;

3. OECD 2007. Guidance on grouping of chemicals. ENV/JM/MONO(2007)28).

 

Conclusions:

EC 234-277-6 has not been tested for repeat dose 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 parental systemic toxicity was proposed for EC 234-277-6

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 ofphosphorodithioic 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.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

supporting 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 nonglandular portion of the stomach

Dose descriptor:

NOAEL

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

Critical effects observed:

no

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

other: EC 224-235-5

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: EC 270-608-0

Reference 6

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

other: EC 224-235-5

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: EC 270-608-0

Conclusions:

No repeat dose study is available for EC 259-048-8. However, sufficient data and information are available to conclude on the potential hazard as well as establish a protective point-of-departure for the risk assessment to ensure safe use. A weight of evidence is provided that demonstrates that additional animal studies are not necessary.

Executive summary:

No repeat dose study is available for EC 259-048-8. However, sufficient data and information are available to conclude on the potential hazard as well as establish a protective point-of-departure for the risk assessment to ensure safe use. A weight of evidence is provided that demonstrates that additional animal studies are not necessary:

1. No repeat dose/target organ toxicity is expected based on:

a. Data for analog substances

b. Low bioavailability based on TK information

c. Low biological reactivity predicted by OECD QSAR toolbox

2. EC 259-048-8 contains an impurity, phenol, dodecyl-, branched (AKA TPP; CAS 121158-58-5), which is a reproductive hazard category 1B. The risk assessment is conducted based on the residual amount of TPP in EC 259-048-8. As a result:

a. The DNEL is based on the extensive toxicity data available for TPP. This is sufficiently protective as demonstrated by comparing the DNELs to an analog zinc dialkyldithiophosphate ester (ZDDP) that has greater bioavailability based on smaller molecular size

b. Appropriate Risk Management Measures are already in place consistent with a reproductive hazard

Reference 7

Endpoint:

sub-chronic toxicity: oral

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

The oral repeat dose toxicity of an analogue of this registration substance, EC 224-235-5, was evaluated in rats at levels up to 500 mg/kg/day according to OECD 407. Treatment-related deaths occurred at 500 mg/kg/day in both sexes. All of these animals had reddened adrenal and pituitary glands and yellow contents in the stomach. Microscopic examinations found edema and inflammation of the stomach in all 500 mg/kg/day group females and thickened mucosa in the nonglandular portion of the stomach in one male. In the 250 mg/kg/day group, one male also had edema and inflammation of the stomach. Clinical signs were toxicologically significant at 250 mg/kg/day and 500 mg/kg/day dose levels. The occurrence of soft stools, diarrhea, mucoid diarrhea, and a decrease in the general condition of the animals was greater than in the controls and lower dose. Significant decreases in body weight gain occurred only in the 500 mg/kg/day group males and females. Food consumption showed a slightly decrease in the 500 mg/kg/day groups although only the food consumption for females was significantly different from the controls. These findings are primary or secondary effects due to the local irritation via the experimental route of administration. Treatment-related increases in mean absolute and relative adrenal gland body weights occurred at the 250 and 500 mg/kg/day dose levels. However, no test article-related histopathological lesions were observed in the adrenal glands to account for the increases. Based on these findings, the systemic and local portal-of-entry no observed adverse effect level (NOAEL) was determined to be 125 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. The RAC decision concerning dodecylphenol, EC No. 310-154-3, for its inclusion on Annex VI (9th ATP) considered long-term systemic effects including repeat dose toxicity.  From this evaluation, it was concluded that the material does not meet the criteria for classification for repeated dose specific organ toxicity 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.

 

Justification for classification or non-classification

In accordance with EU CLP (Regulation (EC) No. 1272/2008) classification of this substance is not required for specific target organ toxicity following repeated exposure.