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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
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:
other: OECD 421 (reproduction/development 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
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 17, 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%
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 O), 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.
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.
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
Duration of test:
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.
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.

Examinations

Maternal 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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: No.
Examinations included:
- Gravid uterus weight: No.
- Number of corpora lutea: No.
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: Yes
- Other:
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: No.
- Skeletal examinations: No.
- Head examinations: No.
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.
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
Historical control data:
No data available.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
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.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
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.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental 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.