N,N,N',N'-tetrakis(2-hydroxypropyl)adipamide

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from Jan. 18, 1994 to Jun. 17, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: N, N, N’, N’-Tetrakis (2-hydroxyethyl) hexanediamide, which is structurally similar to N,N,N',N'-tetrakis(2-hydroxypropyl)adipamide. Thus, the guideline study with GLP is used for read-across to avoid duplicate tests.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Primid XL-552

IUPAC Name:

Primid XL-552

Test material form:

other: white solid

Details on test material:

The sample of test article used in this study was a white solid containing 87.2% Monomer and 2.4 % Dimer. The sample was identified as Toxicology Department Number (TD No.) 93-080; Lot No. 2A-20658

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test material:
One hundred thirty male and one hundred thirty female rats (Crl:CD®BR, 21 days old, non-litter mates) were received from Charles River Laboratories, Inc., Kingston Facility, Stone Ridge, NY on December 28, 1993.
Upon arrival, all animals were examined for physical abnormalities or signs of ill health, and acclimated to the study room for 21 days.
Quarantine procedures of the Laboratory Animal Service Unit were followed for the first 14 days of the acclimation period.
After acclimation, a unique identification number was tattooed onto the tail of each rat. After randomization into treatment groups, each rat received a cage card indicating animal number, group number, dose level, test substance, sex, and protocol number.
HUSBANDRY
Animal husbandry procedures of the Laboratory Animal Service Unit were followed throughout the study. Upon arrival, the rats were placed into a sanitized room. During the first week of the acclimatisation period, animals were housed two (same sex) per cage. Thereafter, animals were individually housed, except during cohabitation (mating).
Males throughout the study, and females during pre-mating and mating, were housed in wire-mesh, stainless steel cages. Suspended above absorbent paper liners these were changed three times/week. During the mating period, cage liners were changed daily. Clean cages were provided at least every two weeks. During gestation and lactation, females were housed individually in polycarbonate cages.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 40 - 60%
- Photoperiod (hrs dark / hrs light):12 hour light/dark cycle

Administration / exposure

Route of administration:

oral: feed

Vehicle:

water

Details on exposure:

DIET PREPARATION
Diets were prepared weekly (2 preparations of 12 kg per dose group) in increasing order of dose. One half of the desired amount of test material was weighed into each of two tared 600 ml Tri-Pour® beakers using a Mettler PM 400 scale. Distilled water was added to the test material until a final volume of 240 ml/beaker was reached. The compound was dissolved using a polytron mixer on low speed.Twelve kilograms of P.M.I Certified Rodent Diet #5002M was weighed using a Sauter E-49 scale, then placed into a Patterson-Kelly Crossflow Mixer equipped with a liquid feed tube and high-speed liquid addition blending bar. The two solutions of test material were added to the blender via the liquid feed tub© while the diet was mixing.
- Storage temperature of food: at room temperature

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 21 days
- Proof of pregnancy: [vaginal plug / 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

Females without a confirmed copulation date at the end of the mating period (21 days) were housed in the same manner as the presumed pregnant females. Females that did not produce a litter within 25 days after separation from the male were presumed non-pregnant and returned to the wiremesh cages until scheduled necropsy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

D. DIET PREPARATION AND SAMPLE ANALYSIS
Group Concentration of test article (ppm) test article required for 12 kg of feed (Q)* Final volume of compound in distilled water (ml)
1 0 0.0 480
2 1000 12.0 480
3 4500 54.0 460
4 20,000 240.0 480
Dietary samples containing test article were analyzed for homogeneity, stability after storage for 7 and 14 days at room temperature, and proximity to target concentrations.
The analytical results confirm that:
1. The procedure used for diet preparation provided homogeneous mixtures of test substance in rodent feed
2. test article was stable in the diet for at least 14 days when stored at room temperature (approximately 68 °F; 20 °C).
3. test article concentrations were within ±10% of nominal target concentrations for all levels, with one exception. A single diet preparation of 1000 ppm was 85.1% of target. Other diet preparations at this concentration were 90 - 92% of target concentrations.
4. Chemical analyses confirm that the nominal concentrations are a satisfactory reflection of the concentrations administered to the animals.

Duration of treatment / exposure:

Exposure period: 11 weeks before the mating and afterwards during the gestation and lactation periods
Pre-mating exposure period (males): 11 weeks
Pre-mating exposure period (females): 11 weeks

Frequency of treatment:

continuously

Details on study schedule:

GID Test Compound DietaryConcentration _Mm)a
Parental Animals/Group
M F Age at Start of Treatment (weeks) Prematingtreatmentperiod(weeks)
1 Control 0 30 30 6 11
2 test group 1000 30 30 6 11
3 test group 4500 30 30 6 11
4 test group 20,000 30 30 6 11

a: based on total product;
M: males; F: females

Necropsy Schedule
Parental Animals: Post-weaning of all litters.
Offspring: Day 4 (culls) and Day 21 postpartum.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

control group

Doses / concentrations 2

Dose / conc.:

1 000 ppm (nominal)

Remarks:

low dose group - equivalent to >50 mg/kg bw/d

Doses / concentrations 3

Dose / conc.:

4 500 ppm (nominal)

Remarks:

mid dose group -equivalent to >225 mg/kg bw/d

Doses / concentrations 4

Dose / conc.:

20 000 ppm (nominal)

Remarks:

high dose group - equivalent to >1000 mg/kg bw/d

No. of animals per sex per dose:

30

Control animals:

yes, concurrent vehicle

Details on study design:

ANIMAL DISTRIBUTION INTO TREATMENT GROUPS
Parental animals were weighed and given a physical examination one week prior to initiation of treatment. One hundred twenty apparently healthy animals of each sex were randomly assigned to one of three treatment groups, or the control group, using a computerized procedure based on body weight. Group body weight means were calculated and statistically analyzed to ensure that no significant differences existed among treatment groups within each sex. Extra animals that were not assigned to a group were returned to Laboratory Animal Services Unit.

Diet preparation records were maintained showing the amount of compound used, feed used, the date of preparation and the date of administration, Uneaten and unused diets were discarded as hazardous waste.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS:
Daily observation for clinical signs

BODY WEIGHT:
Individual body weights for each F0 animal were determined weekly.

FOOD CONSUMPTION:
The food consumption rate was recorded weekly. Fresh diet was added to the jars every week.

Oestrous cyclicity (parental animals):

the morphology of the vagina and uterus during the estrus cycle

Sperm parameters (parental animals):

not examined

Litter observations:

Upon completion of delivery (Day 0 postpartum or Day 0 PP), the number of live, dead, stillborn or uncertain offspring were recorded. Non-viable offspring were classified as stillborn if their lungs did not float in water. An uncertain status was assigned if viability at birth and/or sex could not be determined. Each pup was examined for external alterations and clinical signs. Throughout lactation, cage-side observations were made twice daily to detect dead or moribund animals. Offspring were individually weighed and examined for signs of ill health, reaction to treatment, and abnormal behavior or appearance on Days 0, 4, 7,14 and 21 PP.

Postmortem examinations (parental animals):

GROSS NECROPSY
-All organs, tissues, and body cavities were examined in the parental animals at the scheduled necropsy, as well as in those animals found dead or sacrificed moribund. Animals found dead were necropsied immediately or refrigerated until a necropsy could be performed. Parental animals were euthanized by an IP injection of Nembutal® anesthesia (1 ml/kg body weight) followed by exsanguination.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were collected from each animal and preserved in 10% neutral buffered formalin for histopathologic examination:
Males: epididymides, prostate, testes, seminal vesicles, coagulating gland
Females: ovaries, uterus, cervix, vagina
All: liver, kidney, pituitary and all tissues with gross changes.
The uteri of females that did not deliver were opened and stained with 10% ammonium sulfide to detect very early resorptions. The number of implantation sites were recorded for all females during the necropsy.
Microscopic examination was performed on all tissues collected from control and high dose group parental animals, as well as animals that died or were sacrificed moribund. Since no treatment-related effects were observed, tissues were not examined at lower dose levels.

Postmortem examinations (offspring):

GROSS NECROPSY
-Pups found dead, and pups culled on Day 4 PP were grossly examined for abnormalities of the abdominal and thoracic cavities. All remaining offspring were euthanized by CO2 asphyxiation at the end of the weaning period (Day 21 PP) and similarly examined for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were collected from each animal and preserved in 10% neutral buffered formalin for histopathologic examination:
Males: epididymides, prostate, testes, seminal, vesicles, coagulating gland
Females: ovaries, uterus, cervix, vagina
All: liver, kidney, pituitary and all tissues with gross changes.
The uteri of females that did not deliver were opened and stained with 10% ammonium sulfide to detect very early resorptions. The number of implantation sites were recorded for all females during the necropsy.

Statistics:

The litter (i.e., proportion of affected fetuses per litter, or litter mean) was used as the experimental unit for the purpose of statistical evaluation (1). The level of significance selected was p<0.05.
a, When the one-way ANOVA was significant, Dunnett's test (5) was used to detect significant differences between each treated group and the control group.
b, When more than 75% ties occurred (i.e., 75% of the litters are unaffected for a particular parameter), then Fisher's Exact test was used in place of the Mann-Whitney U test to detect significant differences between each treated group and the control group.

Reproductive indices:

The following reproductive parameters were evaluated statistically:
Male Mating Index (%) = Number of males that mated/Number of males used for mating x 100

Female Mating Index (%) = Number of females mated /Number of females used for mating x 100

Male Fertility Index (%) = Number of sires/Number of males mated x 100

Female Fertility index (%) = Number of pregnant females/Number of females that mated x 100

Gestation Index (%) = Number of females producing litters with at least one live dud / Number of pregnant females x 100

Offspring viability indices:

Viability Index (%) = Number of pups/litter alive on Dav 4.PP/ Number of pups/litter born alive x 100
Lactation index (%) = Number of pups/litter alive at weaning (Dav 21 PP) / Number of pups/litter alive after culling (Day 4 PP) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant decrease in mean body weight was observed in males exposed to 4500 ppm test article during weeks 6 - 12 of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant decrease in mean body weight was observed in males exposed to 4500 ppm test article during weeks 6 - 12 of treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: Statistically significant decreases in feed consumption were observed in males exposed to 1000 ppm during week 19, and in the 4500 ppm dose group during weeks 9, 19, and 20.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

A: in life observation
a. Mortality and Clinical Observations: There was no treatment-related mortality in animals of either sex at doses up to and including 20,000 ppm.
One male in the 4500 ppm treatment group was euthanized for humane reasons during week 12. One female in the 1000 ppm treatment group was found dead following a prolonged delivery. One female in the 1000 ppm group was sacrificed moribund during lactation following weight loss, clinical signs of ill health, and the death of her entire litter. Another female in the 1000 ppm treatment group was sacrificed for humane reasons on Day 29 of gestation because of Lymphosarcoma. These findings were not considered treatment-related due to their spurious occurrences, and lack of a dose-response.
There were no treatment-related clinical signs of toxicity in male or female animals at doses up to and including 4500 ppm.
No treatment-related clinical signs of toxicity were observed in male animals exposed to 20,000 ppm throughout the pre-mating period. However, subsequent to mating soft and/or irregular feces were observed in 24 of 30 animals (note that abnormalities of the feces and urine were not recorded during mating since they could not be ascribed to the male or female animal). Similar findings were observed in only 1 of 30 males in the control group. Since this condition did not develop until after more than 11 weeks of continuous treatment, its toxicological significance and relationship to treatment are unclear.
In females exposed to 20,000 ppm, soft and/or irregular feces were noted in 11 of animals, only during the premating period. In most of these animals, these signs did not develop and persist until after 6.7 weeks of treatment. Only 1 of 30 control animals exhibited similar findings. No clinical signs of toxicity were observed in female animals during gestation or lactation. However, due to housing conditions (use of bedding), abnormalities of feces or urine, unless extreme, could not be readily observed in females during these periods.
b. Body Weight Effects: There were no treatment-related effects on mean body weights in male animals at doses up to and including 20,000 ppm. A statistically significant decrease in mean body weight was observed in males exposed to 4500 ppm test article during weeks 6 - 12 of treatment. Due to the lack of dose-response, this finding was not judged to be treatment-related.
c. Feed Consumption: There were no treatment-related effects on feed consumption in animals of either sex at doses up to and including 20,000 ppm.
Statistically significant decreases in feed consumption were observed in males exposed to 1000 ppm during week 19, and in the 4500 ppm dose group during weeks 9, 19, and 20. Due to the sporadic occurrences and minimal nature of these findings (<10% reduction), and the lack of a dose-response, these reductions were not judged to be treatment-related.
d. Compound Intake: The mean compound intake for all adult animals was calculated from feed consumption and body weight data as below table 1.
Reproductive results:
a. Male Reproductive Indices
There were not treatment-related effects on the male mating index or male fertility index at any dose level.
b. Female Reproductive Indices
There were no treatment-related effects on the female mating index, fertility index or gestation index. Only a single female, in the 1000 ppm dose group, did not survive delivery. This effect was not judged to be treatment-related.
There was no treatment-related effect on the length of gestation. The number of litters with stillborn pups was not affected by test article exposure. There were no litters that were entirely stillborn at any dose level. In only two litters (both in the 4500 ppm dose group) did the entire liveborn litter die, both prior to postnatal day 4. This was not judged to be treatment-related due to the lack of a dose-response.
B. GROSS AND HISTOPATHOLOQICAL FINDINGS: PARENTAL ANIMALS
Dietary administration of 20,000 ppm Primid XL-552 to male and female rats for approximately 10 weeks prior to mating and throughout the mating, gestation and lactation periods did not cause microscopic alterations in the tissues examined. In particular, male and female reproductive tracts were unaffected.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

20 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

a. Clinical observations: No treatment-related signs of ill health were noted in offspring of animals at any dose level.
b. Postnatal Survival and Pup: There were no treatment-related effects on postnatal survival, viability index, or lactation index at any dose level. There were no treatment-related effects on number of liveborn pups/Iitter or number of live pups/litter on days 4, 7, 14 or 21 PP. No treatment-related effects on mean pup weight at birth, or on days 4, 7, 14 or 21 PP were noted. Sex ratio was not altered at any dose level.
c. Gross necropsy: There were no treatment-related findings observed during gross necropsy of offspring at cutting (Day 4 PP) or at weaning (Day 21 PP).

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Generation:

F1

Effect level:

20 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: The mean compound intake for all adult animals:

F-P: females during the premating period;F-G: females during gestation;F-L: females during lactation.

Note that the increase in daily compound intake during the lactation period in all treatment groups is attributable, in part, to consumption of treated feed by both dams and their offspring.

Dietary Concentration (ppm)	Sex	Compound Intake (mg/kg/day)±SE
1000	M	62.6±4.90
	F-P	85.9±5.11
	F-G	69.5±1.01
	F-L	122.6±19.11
4500	M	284.2±22.25
	F-P	387.5±25.00
	F-G	294.1±6,83
	F-L	562.6±61.68
20,000	M	1280.4±100.58
	F-P	1726.7±105.39
	F-G	1380.5±14.71
	F-L	2735.2±408.87

Exposure of rats to test article from approximately six weeks of age, into adulthood, and through production of one generation of offspring had the following results:

1000ppm
Compound Intake:	Males:              62.6 mg/Kg/day Females:Premating:    85.9 mg/kg/day Gestation:  69.5 mg/kg/day Lactation:  122.6 mg/kg
Parental animals:	No treatment-related effects
Reproductive effect	No treatment-related effects
4500ppm
Compound Intake:	Males:            284.2 mg/kg/day Females:Premating:    387.5    mg/kg/day Gestation:  294.1    mg/kg/day Lactation:  562.6    mg/kg/day
Parental animals:	No treatment-related effects
Reproductive effects:	No treatment-related effects
20,000ppm
Compound intake:	Males: 1280.4 mg/kg/day Females:Premating: 1726.7 mg/kg/day Gestation: 1360.5 mg/kg/day Lactation: 2735.2 mg/kg/day
Parental animals:	Soft and/or irregular feces in both sexes. These signs were considered equivocal due to their delayed onset (females: 6-7 weeks; males: 11-14 Weeks.
Reproductive effects:	No treatment-related effects

 

 

Applicant's summary and conclusion

Conclusions:

No treatment related reproductive toxicity was observed when rats were exposed to test article in the diet at concentrations up to and including 20,000 ppm (>1000 mg/kg/day).

Executive summary:

The purpose of this study was to investigate the effects of test article on growth, development and reproductive performance in male and female rats through production of one generation of offspring.

Based on the results of a preliminary range-finding study in male rats, a limit concentration of 20000 ppm (target dose of 1000 mg/kg) test article was selected as the high dose level for the reproductive study. Rats were exposed to test article in the diet at concentrations of 0, 1000, 4500 and 20,000 ppm beginning at approximately six weeks of age. Animals were mated after eleven weeks of exposure; treatment continued throughout gestation, lactation, and until terminal necropsy. No treatment-related effects on mortality, body weight or feed consumption were observed in either sex.

There were no treatment-related effects on any reproductive endpoints including number of litters produced, gestation length, mating and fertility indices (in both sexes), and gestation index. There were no treatment-related effects on number of live born or stillborn pups per litter, postnatal survival viability and lactation indices or growth. No treatment-related gross findings were observed in the parental animals or offspring at any dose. In addition, no treatment-related microscopic changes were observed in the organs examined from parental animals treated with 20,000 ppm. At 20,000 ppm, indications of toxicity were limited to soft and/or irregular feces in both sexes. Based on the available data, it is concluded that reproductive toxicity was not observed at doses up to and including 20,000 ppm.