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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
three-generation reproductive 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:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study has been extensively conducted, but has no histology.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
other: Crl: CD(SD) BR purchased as weanlings
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts (Portage, Michigan facility)
- Age at study initiation: (P=F0) x approximately 7 wks; (F1) x in utero
- Fasting period before study: No
- Housing: individually in suspended, stainless steel, screen bottom cages, held on racks, with
deotized animal cage boards (DACB, Shepherd Specialty Papers, Inc., Kalamazoo, Michigan)lining
the urine- and feces-collecting pans.
During mating periods, double-sized stainless steel cages were used to house one male and one
female rat. Cage boards were changed at least twice weekly and animals were transferred to clean
cages at least biweekly except during the mating period. Pregnant rats (during the last week of
gestation) and rats with young, as well as females that did not exhibit evidence of positive mating,
were individually housed in plastic cages fitted with water bottles filled with tap water. A bedding
material consisting of heat-treated hardwood chips (Beta-Chip, Northeastern Products Corporation,
Warrensburg, New York) covered the bottom of the cages, and was changed at least once
weekly. Variations from these procedures are documented in the raw data.
- Diet : ad libitum from glass containers that limit spillage and contamination and allow easy
inspection of the amount and condition of the feed. The basal diet for this study was ground Purina
Certified Rodent Chow #5002. A record of all lot numbers is included in the raw data. This diet has
been analyzed by the manufacturer for nutritional components and environmental contaminants,
and the minimum or maximum concentrations of these materials are specified.
- Water : partially demineralized by reverse osmosis and sterilized by ultraviolet light, ad libitum
from an automatic watering system (Systems Engineering, Palo Alto, California), except during the
last week of gestation and during lactation when tap water was provided in water bottles to
animals in plastic cages. Hazleton Laboratories America, Inc. (HLA) analyzed the tap water and
water from the automatic system quarterly for total dissolved solids, conductivity, heavy metals,
organophosphates, chlorinated hydrocarbons, microbiological content and selected elements.
Results of these analyses were within normal limits and are on file at HLA.
- Acclimation period: at least 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature : 72°F±3°
- Humidity (%):50%±20%
- Air changes (per hr): at least 10 changes an hour, filtered 100% outside air
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: feed
Vehicle:
acetone
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Approximately 1500g of the weighed rodent chow was transferred to a Hobart N50. DSS was added to a measured amount of acetone and mixed with the basal diet in the Hobart N50 for 5 minutes. This premix was then added to the rodent chow in the Hobart H600T mixer and mixed for 15 minutes to achieve the proper concentration in the feed. Each dose level for the study was prepared independently, first the low dose and then progressively higher doses.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Certified Purina Rodent Chow® #5002
- Storage temperature of food: mixed feed was stored refrigerated in air-tight polyethylene buckets.

VEHICLE
- Justification for use and choice of vehicle (if other than water): acetone
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum 21 days
- After 21 days of unsuccessful pairing females were placed in a plastic cage
- Further matings after two unsuccessful attempts: not applicable
- Proof of pregnancy: vaginal plug/sperm in vaginal smear referred to as day 0 of gestation

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the prepared diets were done prospectively (i.e., before feeding to the animals) using the analytical method in Appendix B. This method is based on the hydrolysis of DSS to 2-ethyl- hexanol which is then quantitated by gas-liquid chromatography. Analyses were performed weekly for the first 4 weeks, weekly during the 2-week premating phase for F0 females, twice during breeding for F1 litters, twice during F1 gestation, twice during F1 lactation, and monthly thereafter.
Because the analytical method for DSS is based on measuring a possible degradation product (2-ethyl hexanol) , it was necessary to determine if measurable quantities of 2-ethyl hexanol were present in nonhydrolyzed diet samples. To do this, several sets of diets were analyzed for DSS and 2-ethyl hexanol after 8 days at ambient conditions and/or after being stored frozen for 1 to 14 days.
Duration of treatment / exposure:
F0 generation animals received the test material at the appropriate dose levels in the diet continuously, from approximately 7 weeks of age (October 4, 1984 for males; November 29, 1984 for females), throughout mating, gestation, and lactation and until they were necropsied on March 4, 5, and 6, 1985.

F1 and F2 generation animals were exposed to the test material in utero, while nursing, continuously from when they weaned to test diets , throughout mating, gestation, and lactation, and until they were necropsied on July 22, 23, and 24, 1985 (F1 animals) and on December 26 and 30, 1985 (F2 animals).
Doses / concentrations
Remarks:
Doses / Concentrations:
0%, 0.1%, 0.5%, and 1.0%
Basis:
nominal in diet
No. of animals per sex per dose:
Dosing (F0, F1 and F2) : 30/sex/group (0%,0.1%,0.5%,1.0%)
Control animals:
yes

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: males: weekly
females: weekly during pre-mating phases; on Days 0, 7, 14,and 20 of
gestation; and on Days 0, 7, 14, and 21 of lactation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined (g ): Yes
- Compound intake, (mg/kg/day): Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice of all adults (F0, F1 and F2): after litters have been weaned
- Organs examined:colon, duodenum, epididymides, (gross lesions), ileum, jejunum, kidneys, liver,
mammary gland with skin, ovaries, prostate, seminal vesicles, stomach, testes, uterus, vagina.


Oestrous cyclicity (parental animals):
Not provided
Sperm parameters (parental animals):
Not provided
Statistics:
Differences between treated groups and the control group were considered statistically significant at p≤0.05 and are indicated with an asterisk(*).
Data for adult animals were analyzed by sex. Body weight; body weight gain; food consumption; reproduction indices; precoital interval; length of gestation; pup viability; body weights, body weight gains, and sex ratios; and litter size (alive and dead by sex) were analyzed separately using one-way nalyzes of variance (ANOVA) with transformation when necessary (log10, square, square root, reciprocal, arcsine rank) to attain variance homogeneity. If the ANOVA was significant, Dunnett’s test was used to compare each treated group with the control. When no transformation could establish variance homogeneity (as indicated by Levene’s test at p<0.01), all data were also examined by nonparametric techniques. These statistics included the Kruskal-Wallis H-test analysis of variance and, if this test was significant, the Nemenyi (F0 and F1 generations) or the Nemeneyi-Kruskal-Wallis (F2 generation) test for multiple comparisons or the Wilcoxon-Mann-Whitney two-sample rank test. Reproduction indices and the total number of live and dead pups were analyzed by the Cochran-Armitage test for trend and Fisher-Irwin exact test for heterogeneity.
Reproductive indices:
Mating = number of females mated( =number of females with vaginal sperm or plug, or
that gave birth to a litter)x100/ number of females placed with males

Female fertility = number of females pregnant(=number of females that gave birth to a litter )x100/
number of females mated

Male fertility = number of males shown to be fertile(=a female giving birth to a litter)x100 /
number of males placed with females

Gestation = number of live litters born x100/ number of pregnant females


Offspring viability indices:
Viability * = (number of pups surviving to Day 4)x100/ number of pups born alive

Weaning+ = (number of pups surviving to Day 21)x100/ number of pups alive at Day 4 postculling

Sex ratio = ( number of male pups )x100/ total number of pups

* Also reported as “Survivability Days 0-4”
+ Also reported as “Survivability Days 4 (postculling)-21”

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment- related clinical observations for F0 males and females. Alopecia was noted with similar frequency in the control and treated groups.A common occurence in rodents maintained on ground feed is malocclusion.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dose levels of 0.5% and 1.0% caused a reduction in body weight for the F0 males and the values were statistically significant at Weeks 9, 10, 18 and 19 for the 1.0% dose level and at Week 17 for the 0.5% dose level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Dose levels of 0.5% and 1.0% caused a reduction in body weight for the F0 males and the values were statistically significant at Weeks 9, 10, 18 and 19 for the 1.0% dose level and at Week 17 for the 0.5% dose level.
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
(number of females pregnant (that gave birth to a litter) / number of females mated)x100
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
(number of males shown to be fertile (defined by a female giving birth to a litter)/number of males placed with females)x100
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Effect level:
0.1 other: %
Based on:
act. ingr.
Remarks:
in the diet
Sex:
male/female
Remarks on result:
other: Generation: maternal/paternal (migrated information)
Dose descriptor:
NOAEC
Effect level:
1 other: %
Based on:
act. ingr.
Remarks:
in the diet
Sex:
male/female
Remarks on result:
other: Generation: offspring (migrated information)

Results: F1 generation

General toxicity (F1)

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights for F1 males and females at the 1.0% dose level were significantly lower than those of controls throughout the premating phase, excluding Week 2 for females, and throughout the treatment period for males.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
DSS administered in the diet to three successive generations of rats at levels of 0.5% and 1.0% caused a reduction in body weights for parental males
of all generations and for F1 and F2 females. Pup weights at the 0.5% and 1.0% dose levels were lower than those of the control in all three generations. However, the reduced body weight did not interfere with growth and development or reproductive performance, and DSS at levels up to 1.0% had no adverse effects on the reproductive function of either sex in any generation.

Based on the results of this study, when DSS was administered in the diet to three succesive generations of rats, the no-observable-effect level (NOEL)
for body weights of parental animals and offspring was 0.1%; the NOEL for reproductive parameters was 1.0% DSS.
Executive summary:

Reproductive toxicity of Docusate sodium was tested in 3-generation toxicity study at dietary dose levels of 0.1, 0.5 and 1.0% in the diet (MacKenzie, 1986). The study was conducted according to OECD 416 and GLP guidelines, and was considered to be reliable, adequate and relevant. The test substance caused a reduction in body weights at the dose levels of 0.5 and 1% in the diet for parental males of all generations and for F1 and F2 females. Pup weights at the 0.5% and 1.0% dose levels were lower than those of the control in all three generations. However, the reduced body weight did not interfere with growth and development or reproductive performance, and the test substance had no adverse effects at levels on the reproductive function of either sex in any generation up to 1%. There were no other effects on parental or reproductive parameters. Based on the results of this study the NOEL for body weights of parental animals and offspring was 0.1%; the NOEL for reproductive parameters was 1.0%, which was considered to correspond with approximately 750 mg/kg bw/day.