Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
14 Feb 1994 - 14 Feb 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study compliant with standardised tests, and is sufficiently detailed. The original study is considered Reliable without Restrictions according to the criteria of Klimisch, 1997. However, for this assessment, since the data were derived for a structually similar material, the reliability is reduced to Reliable with Restrictions according to the criteria of Klimisch, 1997.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Name of test material (as cited in study report): Zinc O,O-diethylhexyldithiophosphate (ZDDP)
- Physical state: clear colorless viscous liquid
- Storage condition of test material: the bulk test article was stored in a sealed container at room temperature and was considered to be stable under these conditions.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
-Source: Charles River Breeding laboratories, Inc, Portage, Michigan.
-Age at study initiation: 10 weeks old (male and female)
-Weight at study initiation: At the start of treatment the males weighed ranged from 320 to 404 g, and the females weighed ranged from 196 to 266 g.
-Fasting period before study: None
-Housing: Upon arrival and until pairing, all animals were housed in clean, wire-mesh cages suspended above cage-board. The animals were paired for mating in the home cage of the male. Following positive identification of mating, the males were housed in individual suspended wire-mesh cages until necropsy. Bred females were transferred to clean, individual plastic maternity cages with nesting material, ground corn cob bedding. The dams were housed in these cages through lactation day 4, the scheduled day of necropsy. Females which did not deliver were necropsied on post coital day 25. Females for which there was no evidence of mating were placed in a clean, plastic maternity cage with nesting material upon completion of a 15-day mating period.
-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, in meal form; the lot numbers used were recorded. The diet utilized at WIL Research Laboratories, Inc., is a certified feed with appropriate analyses performed and provided by the manufacturer.
-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): 17.8 to 23.8 oC
-Humidity (%): 24% to 72%
-Air changes: no data available.
-Photoperiod: twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: February 17t, 1994 To: April 7, 1994.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test material, ZDDP, was weighed for each dose group into tared precalibrated storage containers. A sufficient amount of vehicle was added to attain an appropriate volume for mixing. A magnetic stir bar was added and the mixtures were stirred continuously throughout the sampling and dosing procedures.
The dosing formulations were prepared weekly and stored at room temperature. The dosing formulations were visually inspected by the study director prior to the initiation of dosing and were found to be homogeneous and satisfactory for administration.
DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

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, 6, 20, 40 mg/ml.
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): NDA
- Purity: 100%
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 15 days
- Proof of pregnancy: [vaginal copulatory plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no.
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: when evidence of copulation was not detected after 10 days of pairing, the female was placed with another male from the same treatment group for an additional five days, with one exception. A female in the 200 mg/kg/day group was not paired with a second male after no evidence of mating occurred following 10 days of pairing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling:
For the batch prepared on the day prior to the first day of dosing (February 17, 1994; week 0), two 5 ml sample aliquots from the control group (middle stratum) and twelve 5 ml aliquots (four aliquots each from the top, middle and bottom strata) from each of the 30, 100 and 200 mg/kg/day groups were collected. Two sets of these samples were sent to the sponsor for homogeneity analysis, while the other two sets were stored at WIL Research Laboratories, Inc. Sixteen 5 ml aliquots (four aliquots each from the middle stratum of each dose level, including the control group) were also collected on February 17, 1994. Two sets of these samples were sent to the sponsor for concentration analysis, while the other two sets were retained at WIL Research Laboratories, Inc.

Beginning on February 24, 1994 (week l), and continuing through April 7, 1994 (week 7), sixteen 5 ml sample aliquots (four aliquots each from the middle stratum of each dose group, including the control group) were collected from each weekly dosing preparation. For each of these preparations, two sets of samples were stored at Wil Research Laboratories, Inc., and two sets were forwarded to the sponsor for concentration analysis. The samples were shipped under ambient conditions.
Samples for 8-day stability analysis were inadvertently not sent to the sponsor as specified in the protocol. Based on the results of the retrospective concentration and homogeneity analyses of the test material formulations performed by the sponsor, no apparent differences between the analyzed and target concentrations of the test material formulations were noted. Thus, the sponsor elected not to retrospectively analyze the samples for 8-day-stability. This deviation was not considered to adversely affect the integrity of the data or the outcome of the study.

Verification of ZDDP Dosing Suspensions-Abstract
Samples of dosing suspensions (Zinc O,O-di-2-ethylhexyldithiophosphate in corn oil) used in an oral reproductive/developmental screening study were analyzed to verify their nominal concentrations. A direct dilution procedure was employed to prepare the samples for elemental analysis by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES). The consistency of the analytical data with the theoretical values of the dosing suspensions used in the concentration portion of the study, with the exception of the lowest dosing level, verifies their nominal concentrations. The dosing suspensions were determined to be homogeneous based on the analytical data gathered.
Duration of treatment / exposure:
Male animals were dosed for at least 14 consecutive days prior to mating and continuing for a total dosing period of 28 days.
Females were dosed for a minimum of 14 days prior to mating and continuing until the scheduled necropsy (lactation day 4 for females that delivered a litter; post-mating day 25 for females that did not deliver a litter).
The concurrent control animals received corn oil on comparable regimens.
Frequency of treatment:
Daily
Details on study schedule:
Pre-mating treatment period: 14 days
Age at mating of the mated animals in the study: ~12 weeks;
Cooccupant period: Up to 10 days with initial male; if positive evidence of mating not present (sperm or copulatory plug) then female paired with a second proven breeder male from the same dose group for up to five additional days.
Male treated for 28 days in total.
Female treated until lactation day 4.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
200 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
-Dose selection rationale: The dose levels were set based on a 28-day toxicity study in the rat. In that study a dose level of 500 mg/kg/day has been demonstrated to exceed a maximum tolerated dose for a subsequent reproduction/developmental toxicity study in rats. A dose level of 10 mg/kg/day was considered to be the NOAEL (no observable adverse effect level) for systemic toxicity.

- Rationale for animal assignment (if not random):
At the conclusion of the acclimation period, all available animals were weighed and examined in detail for physical abnormalities. At the discretion of the study director, animals judged to be in good health and meeting acceptable body weight requirements were selected for use in the computer randomization procedure. At this time, the individual body weights and corresponding animal identification numbers were entered into WIL's Toxicology Data Management System. A printout containing the animal numbers, corresponding body weights and individual pup assignment was generated based on body weight stratification randomized in a block design. The animals were then arranged into groups according to the printout. The experimental design consisted of three treatment groups and a control group, with 12 males and 12 females per group. Individual body weights per group at randomization were within ± 20% of the group mean body weight for each sex. The males were approximately 10 weeks old at the initiation of dosing and body weights ranged from 320 g to 404 g on the first day of treatment. At the initiation of treatment, females were approximately 10 weeks old and body weights ranged from 196 g to 266 g. Sixteen females were not within the protocol specified weight range (200-225 g). However, these animals were within the specitid age range (70- 80 days). These deviations in weight had no apparent effect on the outcome of the study.


Positive control:
no data available

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were observed twice daily for appearance, behavior, moribundity and mortality.
- Cage side observations checked in table [No.?] were included. No table was included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were recorded weekly throughout the study period for the males and the females. Males and females were also observed at the time of dosing and one hour following dose administration. On the first day of dosing, the parental animals were also observed at 2 and 4-hours following dose administration. All significant clinical findings were noted at the post- dosing observations. The observations included, but were not limited to, changes in the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system function, somatomotor activity and behavior patterns. Females which delivered were also observed twice daily during the period of expected parturition and at parturition for dystocia.
BODY WEIGHT: Yes
- Time schedule for examinations: individual F0 male body weights were measured weekly -and are presented for weeks 0-4. Mean body weights were calculated for each of these periods. Corresponding weekly body weight changes were also calculated for each weekly interval.
Individual F0 female body weights were measured weekly, beginning with the initiation of treatment and continuing until evidence of copulation was observed, and are presented for weeks 0-2 (the last recorded weekly body weight of all females prior to pairing). Mean body weights were calculated for each of these weeks. Mean body weight changes were calculated for each weekly interval. Once evidence of mating was observed, female body weights were measured on gestation days 0, 7, 14 and 20, and on lactation days 1and 4. Mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding gestation or lactation interval.
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 F0 male and female food consumption was measured weekly until the initiation of the mating period. Food intake was not recorded during the mating period. Once evidence of mating was observed, individual female food consumption was measured on gestation days 0, 7, 14 and 20, and on lactation days 1 and 4. Food intake was calculated as g/animal/day and g/kg/day for the corresponding body weight change intervals.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available.
- Time schedule for examinations: No data available
Oestrous cyclicity (parental animals):
No data available.
Sperm parameters (parental animals):
Parameters examined in F0 male parental generations:
testis weight: yes
epididymis weight: yes
daily sperm production: no data available.
sperm count in testes: no data available.
sperm count in epididymides: no data available.
enumeration of cauda epididymal sperm reserve: no data available.
sperm motility: no data available.
sperm morphology: no data available.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [no]
- If yes, maximum of [no.] pups/litter ([equal sex distribution as nearly as possible); excess pups were killed and discarded: not applicable.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[litter viability and deaths, viability index, clinical observations, body weights, sex determination, and live litter size]

GROSS EXAMINATION OF DEAD PUPS:
[yes for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [The F0 adults were killed after treated for 28 days.]
- Maternal animals: All surviving animals [The F0 females from each dose group were allowed to deliver naturally and rear their young to post-natal day 4.]

GROSS NECROPSY
- Gross necropsy consisted Coagulating gland, Testes with epididymides and Vas Deferens, Ovaries and oviduct (2), Pituitary, Uterus with vagina, Prostate, All gross lesions, Seminal vesicles

HISTOPATHOLOGY: Epididymides, Prostate, Cervix, Seminal vesicles, Coagulating gland, Testes, Ovaries, Uterus, Pituitary gland, vagina, Vas Deferens, all gross internal lesions
ORGAN WEIGHTS: Brain, Kidneys, Liver, Ovaries, Pituitary, Testes, Epididymides
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age: not applicable.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: all viable pups were necropsised on lactation day 4.
GROSS NECROPSY
- Gross necropsy consisted of stomach, blood vessel, spleen, and pancreas.

HISTOPATHOLOGY / ORGAN WEIGTHS
No data available.
Statistics:
All analyses were conducted for a minimum significance level of 5% comparing each treated group to the vehicle control group; all means are presented with
standard deviations. All tests for significance at the 5% probability level were two-tailed for the group comparisons. Data obtained from nongravid animals were excluded from statistical analysis following the mating period. The litter was used as the experimental unit. Chi-square test correction fact with Yates correction factor was used for Pup Sex Ratios, Pup Survival Indices, Mean No. Stillborn and Dead Pups, Parental Fertility Indices; ANOVA (two tailed) with Dunnett's - test was used for F0 Body Weights and Weight Gains, Gestation and Lactation Body Weights and Weight Gains, Parental Food Consumption, Mean Litter Weights, Length-of Gestation, Live Litter Sizes, Organ Weights; Kolmogorov-Smimov (one-tailed) test was used for Histopathological Findings.
Reproductive indices:
Female Mating Index (%) = number of females with determined copulations / total number of female used for mating x 100

Male Mating Index (%) = number of males with evidence of mating / total number of male used for mating x 100

Female Fertility Index (%) = number of pregnant females / total number of females used for mating x 100

Male Fertility Index (%) = number of male siring at least 1 litter / total number of males used for mating x 100
Offspring viability indices:
Live Litter Size = total viable pups day 0/ number litters with vialble pups day 0 x 100

Viability Index (%) = pups viable on day 1 or 4 / pups viable on day 0 x 100.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

-CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Five animals in the 200 mg/kg/day group died prior to the scheduled necropsies. Male nos. 22066 and 22084 in this group died on study days 12 and 19, respectively. Female nos. 22112, 22127 and 22115 in this group died on study days 8, 27 and 39, respectively. Clinical signs noted for at least two of these animals at the daily examinations, at the time of dosing and/or I-hour post dosing on the day of death or the day prior to death were staining, matting or material (brown, tan, yellow, red and/or clear) on various body surfaces, respiratory distress (rales, gasping, labored respiration), hunched appearance and yellow mucoid diarrhea. Single occurrences of unkempt appearance, body cool to touch and aggressiveness were also noted in these animals prior to death. Two females in the 100 mg/kg/day group (nos. 22142 and 22147) and one female in the 200 mg/kg/day group (no. 22130) were euthanized on lactation days 1 or 2 due to total litter loss. Clinical signs observed in these females prior to euthanization were similar to those noted in the animals that died. These findings included staining, matting or material (brown, red, tan and/or clear) on various body surfaces, respiratory distress (rales, gasping) and yellow mucoid diarrhea. In addition, female no. 22147 in the 100 mg/kg/day group exhibited signs of dystocia on lactation day 0, and female no. 22130 in the 200 mg/kg/day group exhibited abnormal nesting behavior on lactation day 1.
All other animals survived to the scheduled necropsies. The predominant clinical signs observed in these animals consisted of the following. Salivation was noted frequently in the 100 and 200 mg/kg/day group males and females at the time of dosing and/or one hour following dosing. Brown staining on various body surfaces was observed frequently one hour following dosing in males and females at dose levels of 100 and 200 mg/kg/day. Other clinical signs apparently related to ZDDP administration were intermittent occurrences of respiratory distress (rales, gasping) and excreta-related findings (mucoid feces, mucoid diihea, diiea andlor soft stool) in the 100 and 200 mg/kg/day group males and females. No clinical signs that could be related to compound administration were observed in the 30 mg/kg/day group males and females.

-BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
a. WEEKLY
Mean weekly body weights in the 200 mg/kg/day group males were comparable to the control group values at weeks 0-4. None of the differences were statistically significant. Mean body weight gains for the 200 mg/kg/day group males during weeks 0-1 and 1-2 were slightly reduced, though not statistically significant, when compared to the control group values. A significantly reduced (p<0.01) mean body weight gain for the 200 mg/kg/day group males occurred during weeks 2-3. Mean body weight gain for the high dose group males continued to be reduced (not significant) during weeks 3-4 in comparison to the control group values. Mean cumulative body weight gain for the 200 mg/kg/day group males from weeks 0 to 4 was significantly reduced (p<0.05) in comparison to the control group value.
Mean weekly body weights, body weight gains and cumulative body weight gains for the 200 mg/kg/day group females prior to breeding (weeks 0-2) were similar to the control group values. None of the differences were statistically significant.
At dose levels of 30 and 100 mg/kg/day, mean weekly body weights for the males were comparable to the control group values at weeks 0, 1, 2, 3 and 4. Mean body weight gains for the 30 and 100 mg/kg/day group males during weeks 0-1, 1-2 and 2-3 were comparable to the control group values. During weeks 3-4, the 100 mg/kg/day group males had a simcantly reduced @< 0.05) mean body weight gain. This reduction was due to male no. 22062 which lost 14 g of body weight during weeks 3-4. A similar decrease was not observed in the 200 mg/kg/day group males and no relationship to treatment was evident. Mean body weight gain for the 30 mg/kg/day group males during weeks 3-4 was comparable to the control group value.
Mean weekly body weights for the 30 and 100 mg/kg/day group females were increased prior to breeding (weeks 0, 1 and 2) in comparison to the control group values. The difference between the control group and the 100 mg/kg/day group females at week 2 was statistically significant (p<0.05). Mean cumulative body weight gain for the 100 mg/kg/day group females was simcantly increased (p <0.05) during weeks 0 to 2 when compared to the control group value; this increase was not considered to be related to treatment. Mean body weight gains for the 30 -and 100 mg/kg/day group females prior to breeding (weeks 0-1 and 1-2) were similar to the control group values. None of the differences were statistically significant.
b. GESTATION
Mean gestation body weights and body weight gains in the 30, 100 and 200 mg/kg/day groups were simitar to or slightly greater than the control group values. No relationship to treatment was apparent.
c. LACTATION
Mean body weights in the 30, 100 and 200 mg/kg/day groups were similar to the control group values on lactation days 1 and 4. None of the differences were statistically significant. Mean body weight gains in these groups were slightly reduced, though not statistically significant, when compared to the control group value. No relationship to treatment was evident.

-TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The test material was administrated via oral gavage.

-REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Estrous cycle regularity was not examined.

- REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No data available.

-REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive performance was not adversely affected by compound administration at dose levels of 30, 100 and 200 mg/kg/day. Male mating indices were. 91.7%, 91.7%, 91.7% and 81.8% and female mating indices were 100%, 91.7%, 100% and 90.9% for the control, 30, 100 and 200 mg/kg/day groups, respectively. Males which did not sire a litter numbered 1, 2, 2 and 4 in the same respective dose groups. In these same dose groups, 0, 1, 1 and 2 females, respectively, had evidence of mating but did not deliver; all females which had evidence of mating and did not deliver were determined to be nongravid at the post mrfem examination, with the following exceptions. Female no. 22115 had evidence of mating but died prior to the expected parturition; this female was determined to be gravid at the post rnonem examination. Female no. 22127 in the 200 mg/kg/day group died on gestation day 5, too early for pregnancy confirmation, and was handled as nongmvid for statistical purposes. Female no. 22096 in the 30 mg/kg/day group had no evidence of mating and was nongmvid.
Fertility indices for males were 91.7%, 83.356, 83.3% and 81.8% and for females were 100%, 83.3%, 91.7% and 81.8% in the control, 30, 100 and 200 mg/kg/day groups, respectively. Although the male and female fertility indices in the 200 mg/kg/day group (81.8%) were slightly lower than the control group values (91.7% and 100.0%, respectively), they were within the range of values in the WIL reproductive historical control data (66.7%-100.0% and 64.0%- 100.0%,respectively). These slight numerical differences were not statistically significant and represented only 1 or 2 fewer successful matings out of a total of 11 or 12 males and females used for mating in the control and high dose groups. Thus, these slightly reduced fertility indices for males and females in the 200 mg/kg/day group were not clearly a treatment related effect.
The mean numbers of days between pairing and coitus in the treated groups were similar to the control group values.

-ORGAN WEIGHTS (PARENTAL ANIMALS)
No adverse effects were apparent on brain, liver, kidney, testes, ovary or pituitary weights in the treated group males and females when evaluated on an absolute basis and relative to final body weight. None of the differences between the treated groups and control group values were significant.

- GROSS PATHOLOGY (PARENTAL ANIMALS)
1. F0 UNSCHEDULED DEATHS
Two males (nos. 22066 and 22084) in the 200 mg/kg/day group died on study days 12 and 19, respectively. Male no. 22066 had a distended stomach. Male no. 22084 was internally normal. Three females (nos. 22112, 22127 and 22115) in the 200 mg/kg/day group died on study days 8, 27 and 39, respectively. Female no. 22112 had a reddened pituitary and yellow contents in the intestinal tract. Female no. 22127 was nongravid (died gestation day 5) and internally normal. Female no. 22115 died on gestation day 21 and had two late resorptions (mummified) and 14 normally developing implantations in utero. A distended and gas filled cecum and stomach and dark red contents or areas in the jejunum and stomach were also noted for this female at the necropsy examination.

2. F0 FEMALE SPOST-MATING DAY 25
One female in each of the 30 and 100 mg/kg/day groups (nos. 22105 and 22113, respectively) had evidence of mating but did not deliver and were necropsied on gestation day 25. Both females were nongravid. Female no. 22105 had a hemorrhagic thymus gland and female no. 22113 was internally normal.

3. F0 FEMALES25 DAYS FOLLOWING THE BREEDING PWOD
Female nos. 22096 and 22118 in the 30 and 200 mg/kg/day groups, respectively, had no evidence of mating and were necropsied 25 days following the conclusion of the breeding period. These females were nongravid and internally normal.

4. F0 FEMALES WITH TOTAL LITTER LOSS
Two females in the 100 mg/kg/day pup (nos. 22142 and 22147) and one female in the 200 mg/kg/day pup (no. 22130) were euthanized in extremis due to total litter loss and were necropsied within 24 hours. Female no. 22142 was internally normal and female no. 22147 had a distended, gas filled and thickened cecum. Female no. 22130 had dark red contents in the stomach.

5. F0 FEMALES LACTATION DAY 4
At the scheduled necropsy on lactation day 4, all females in the control, 30, 100 and 200 mg/kg/day groups were internally normal.
The calculated differences between the number of pups born and the number of implantation sites counted at the scheduled necropsy in the treated groups were comparable to the control group values. None of the differences were statistically significant.

6. F0 MALE SCHEDULED NECROPSY
At the scheduled necropsy of F, males, no compound-related internal findings were observed at any dose level. Male no. 22067 in the 30 mg/kg/day group had dark red lungs and red foamy contents in the trachea. AU other animals were internally normal.

-HISTOPATHOLOGY (PARENTAL ANIMALS)
No microscopic lesions attributed to ZDDP administration were observed in any tissues upon histopathological examination. The frequency of lesions observed in the treated group males and females was similar to that observed for the control group or the findings were noted in single animals. Microscopic lesions observed in the females which had total litter loss, such as hyperplasia and hypertrophy of the uterus vessels and/or cytoplasmic vacuolation of the vagina, were not considered to be related to treatment.
Microscopic examination of gross lesions noted at the scheduled necropsy did not reveal any effects of ZDDP administration.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Sex:
male/female
Basis for effect level:
clinical signs
mortality
other: gastric irritation

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
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:
effects observed, treatment-related
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Viability indices on lactation days 1 and 4 remained above 99% in the control and 30 mg/kg/day groups. Viability indices for the 100 mg/kg/day group at lactation days 1 and 4 were decreased (85.9% and 84.4%, respectively), when compared to the values observed in the WIL reproductive historical control data (96.9%and 96.0%, respectively). Both of these values were significantly decreased (p<0.01) when compared to the control group values. Viability indices at lactation days 1and 4 for the 200 mg/kg/day group were 98.2% and 86.2%, respectively. The lactation day 4 value was significantly lower (p<0.01) than the control group value. The decreased viability indices in the 100 and 200 mg/kg/day groups were due primarily to the females in these groups which had total litter loss.

CLINICAL SIGNS (OFFSPRING)
On lactation day 0 and post-natal days 1-4, pups were found dead in the control, 30, 100 and 200 mg/kg/day groups. Two pups in each of the 100 and 200 mg/kg/day groups were missing and presumed cannibalized. Total litter loss was noted for female nos. 22142 (12 pups) and 22147 (15 pups) in the 100 mg/kg/day group and female no. 22130 (14 pups) in the 200 mg/kg/day group.

BODY WEIGHT (OFFSPRING):
No adverse effects on mean pup weights were apparent on lactation days 1 and 4 at dose levels of 30, 100 and 200 mg/kg/day. None of the differences between the treated groups and control group values were statistically significant.

SEXUAL MATURATION (OFFSPRING)
No data available.

ORGAN WEIGHTS (OFFSPRING)
No data available.

GROSS PATHOLOGY (OFFSPRING)
An increased incidence of pups with no milk in the stomach was noted in the 100 mg/kg/day group. The only other remarkable necropsy finding for pups found dead during the post-natal period was a heart and great vessel anomaly for pup no. 22097-01 in the 200 mg/kg/day group. This malformation consisted of the pulmonary arteries arising from the ascending aorta, the brachiocephalic trunk and left carotid arising from the pulmonary trunk, an absent ductus arteriosis and a small opening in the anterior portion of the septum. Thirteen pups in the 200 mg/kg/day group were noted with body cool to touch. Other observations on the general physical condition of the F,pups in the treated groups occurred at a low incidence, similarly in the control group and/or in a non dose-related manner.

At the post-natal day 4 scheduled necropsy, internal findings (with the exception of presence of milk in the stomach) were noted for 1, 1 and 3 pups in the 30, 100 and 200 mg/kg/day groups, respectively. Pup no. 22106-04 in the 30 mg/kg/day group had unilateral microphthalmia. Pup no. 22148-08 in the 100 mg/kg/day group had a major blood vessel variation, consisting of the right carotid and right subclavian arising independently from the aortic arch. Pup no. 22123-12 in the high dose group had situs inversus. A filamentous tail was noted for pup no. 22146-16 in the 200 mg/kg/day group. The spleen was attached to the left kidney and multiple white foci on the pancreas were noted for pup no. 22097-06 in the 200 mg/kg/day group. No other remarkable internal findings were noted at any dose level at the scheduled necropsy.

HISTOPATHOLOGY (OFFSPRING)
No data available.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day
Sex:
male/female
Basis for effect level:
clinical signs
mortality

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

F0 (Parental Generation)

Two males and three females in the high dose group (200 mg/kg/day) died prior to scheduled necropsy (test days 12, 19, 8, 27 and 39). These deaths were considered treatment related. Two females in the mid dose group (100 mg/kg/day) and one female in the high dose group were euthanized on lactation days 1 or 2 due to total litter loss. Five of these animals exhibited gastric irritation upon necropsy. All other animals survived to their scheduled sacrifice.

Clinical signs noted in the Found dead or sacrificed animals included staining, matting of fur, respiratory distress, hunched appearance and mucoid diarrhea. Clinical findings noted for the surviving mid and high dose males and females included post dosing salivation, brown staining, respiratory distress and diarrhea. No treatment related clinical findings were observed in the low dose (30 mg/kg/day) animals.

 

Fertility indices (%) for the high dose males and females were slightly lower then control as follows:

                 Control            30 mg/kg       100 mg/kg         300 mg/kg

Males           91.7                  83.3                83.3                   81.8

Females      100                    83.3                91.7                   81.8

These values were within the range of the test facility historical control data (64-100%). In addition, differences from control were not statistically significant and represent only 1 or 2 fewer successful matings out of 11 or 12 males or females used for mating in the control and high dose groups. A microscopic examination of the reproductive organs of these animals did not reveal any treatment-related effects. The Study Director concluded that the low and mid dose groups were unaffected and that these data did not clearly reflect a treatment related effect in the high dose group. Other reproductive parameters (mating indices, days between pairing and coitus, gestation length and parturition) were unremarkable in all treated groups.

 

The premating (weeks 1-4) mean body weight gain of the high dose males was statistically significantly reduced compared to control. The mean body weights of the low and mid dose males and all treated female groups were unremarkable during the premating period. Gestation and lactation body weights were unremarkable in all treated groups. Food consumption data were unremarkable in all treated groups (males and females) during the premating, gestation and lactation periods. With the exception of the gastric irritation noted above in several unscheduled deaths, the macroscopic data were unremarkable. Absolute and relative (to body weight) organ weight data as well as the microscopic examination data of the F0 males and females were unremarkable. There were no treatment-related findings evident in any of these data.

 

 

F1 Litter Data

Pup body weights, live litter size and sex ratios were unremarkable. No treatment related effects were evident. An increased number of dead pups was noted in the mid dose group on day 0 of lactation.   Pup viability indices in the mid dose (lactation days 1 and 4) and high dose (lactation day 4) groups were reduced. This was attributed to total litter loss by three females. Increased pup deaths were observed in the mid and high dose groups during the post-natal period. An increased incidences of pups without milk in the stomach was noted in the mid dose group. No treatment related effects were evident in the necropsy data of these found dead pups or in the necropsy data from scheduled pup necropsies.

 

Chemical analysis of dosing suspensions confirmed that they were homogeneous and of appropriate concentration throughout the study.

Applicant's summary and conclusion

Conclusions:
Parental (F0) toxicity was exhibited at dose levels of 100 (mortality, clinical findings) and 200 mg/kg/day (mortality, clinical signs, reduced body weight gain, gastric irritation). A slightly reduced fertility index was also observed at 200 mg/kg/day. No F0 toxicity was observed at 30 mg/kg. Neonatal (F1) toxicity (mortality) was observed at 100 and 200 mg/kg/day. No F1 toxicity was observed at 30 mg/kg. Based on these findings the Study Director concluded that the NOAEL for both parental and neonatal toxicity was 30 mg/kg/day.
Executive summary:

This screening study was designed to determine the potential adverse effects of Zinc O,O-diethylhexyldithiophosphate(ZDDP) on male and female reproduction in rats. The test material was administered orally by gavage to three groups of 12 F0 male and 12 F0 female Sprague Dawiey Crl:CD®BR rats. Dosage levels were 30, 100 and 200 mg/kg/day. For comparative purposes, a control pup of identical design was concurrently dosed with Mazola®corn oil on a comparable regimen. A dose volume of 5 ml/kg was used in all dose groups. All F0 animals were dosed for a minimum of 14 days prior to mating and through the day of necropsy for each F, animal. AU animals were observed twice daily for appearance and behavior. Body weights were recorded weekly for both sexes prior to mating; maternal body weights were also recorded on gestation days 0, 7, 14 and 20 as well as lactation days 1 and 4. Food consumption was measured for corresponding intervals prior to mating, during gestation and during lactation. AU of the surviving F0 females were allowed to deliver and rear their pups to lactation day 4. The offspring were also potentially exposed in urero and through nursing during lactation days 1-4 until euthanization on post-natal day 4. The surviving F, dams were necropsied on lactation day 4, following at least 43 days of dosing. The surviving F0 males were necropsied after the breeding period, following 28 days of dosing. F0 females with total litter loss were necropsied within 24 hours. F0 females which failed to deliver were necropsied on post-mating day 25 (evidence of mating) or 25 days following the breeding period (no evidence of mating). Organ weights were collected (all F0 treated groups) and microscopic examinations were conducted (control and high dose groups and all parental animals not surviving to the scheduled necropsies).

Slightly reduced fertility indices for males and females in the 200 mg/kg/day group (81.8% compared to control values of 91.7% and 100%, respectively) were not clearly related to treatment. Fertility indices in the 30 and 100 mg/kg/day groups were unaffected by treatment. Other reproductive parameters (mating, days between pairing and coitus, gestation and parturition) were unaffected by treatment at all dose Levels.

Two males and three females in the 200 mg/kg/day group died prior to the scheduled necropsies. Two and one females in the 100 and 200 mg/kg/day groups, respectively, were euthanized due to total litter loss prior to the scheduled necropsy. Five of these animals had internal changes consistent with gastric irritation. All other animals survived to the scheduled necropsies. The predominant clinical findings noted for surviving males and females in the 100 and 200 mg/kg/day groups at the time of dosing and/or 1-hour post dosing were salivation and brown staining on various body surfaces. Other treatment-related clinical signs observed for the 100 and 200 mg/kg/day group males and females were respiratory distress (rales, gasping) and excreta-related findings (mucoid feces, mucoid diarrhea, diarrhea and/or soft stool). No clinical signs related to ZDDP administration were observed in the 30 mg/kg/day group males and females.

Reduced mean body weight gains occurred in the 200 mg/kg/day group males during weeks 0-1, 1-2, 2-3 and 3-4. Weekly body weight gains in the 30 and 100 mg/kg/day group males and the 30, 100 and 200 mg/kg/day group females were not adversely affected by treatment. Gestation and lactation body weight gains in the 30, 100 and 200 mg/kg/day group females were not adversely affected by treatment. Food consumption in the 30, 100 and 200 mg/kg/day group males and females was unaffected by treatment.

No compound-related internal findings were noted in the surviving animals in the treated groups at the necropsy and microscopic examinations. Microscopic examination of gross lesions noted at the scheduled necropsies did not reveal any adverse effects of ZDDP administration. No adverse effects were apparent on brain, liver, kidney, testes, ovary or pituitary weights in the treated F,, males and females.

An increased number of dead pups on lactation day 0 was noted in the 100 mg/kg/day group. Pup viability indices for the 100 mg/kg/day group (lactation days 1 and 4) and the 200 mg/kg/day group (lactation day 4) were reduced (due to the three females with total litter loss). Increased numbers of pups in the 100 and 200 mg/kg/day groups were found dead during the post-natal period. The predominant clinical observation noted for the 200 mg/kg/day pups was body cool to touch. F, pup sex ratios were not adversely affected by compound administration at any dose level. Necropsy findings did not reveal a cause of death for the F, pups that died or indicate a relationship to treatment for the F, pups that were euthanized on post-natal day 4.

 

In conclusion, parental toxicity was exhibited at dose levels of 100 and 200 mg/kg/day by mortality and clinical signs. Parental toxicity was also exhibited at the 200 mg/kg/day dose level by inhibition of body weight gain in males and signs of gastric irritation. No parental toxicity was observed 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. Neonatal toxicity (mortality) in the F1 generation was observed at the 100 and 200 mg/kg/day dose levels. Neonatal toxicity was also noted at the 200 mg/kg/day dose level 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.