Hexanediamide, N1,N1,N6,N6-tetrakis(2-hydroxyethyl)-

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1994, January 17 to 1994, December 27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well described GLP compliant study conducted according to recognized international test guidelines.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

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:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Primid XL-552

IUPAC Name:

Primid XL-552

Details on test material:

The sample of Primid XL-552 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 N0.) 93-080; Lot No. 2A-20658

Test animals

Species:

rat

Strain:

other: Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The rats were received from Charles River Laboratories, Inc; Kingston Facility, Stone Ridge NY on December 28, 1993
- Age at study initiation: 6 weeks old
- Housing: during the first week of acclimation period, animals were housed two (same sex) per cage. Thereafter, animals were individually housed, ex cept during cohabitation (mating). Males throughout the study, and females during premating and mating, were housed in wire-mesh, stainless steel cages. (13.5 in x 7 in x 7 in; 34.3 cm x 17.8 cm x 17.8 cm). During gestation and lactation, females were housed individually in polycarbonate ca ges (21 in x 11.5 in x 8 in; 53.3 cmx 29.2cm x 20.3 cm).
- Diet: animal were fed ad libitum with P.M.I. Certified Rodent Diet #5002M
- Water: Filtered tap water was available ad libitum via an automatic watering systam. Femals during gestation and lactation received water ad libitum via a water bottle.
- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 73°F (22.8°C)
- Humidity (%): the relative humidity range was about 40-60%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle was matained during the study.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Group Concentration Primid XL-552 required Final volume of compound
of Primid XL-552 (ppm) for 12Kg of feed (g) in distilled water (ml)
1 0 0.0 480
2 1000 12.0 480
3 4500 54.0 480
4 20.000 240.0 480

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 materials was weighed into each of two beakers. Distilled water was added to the test material until a final volume of 240 ml/beakers was reached. The compound was dissolved using a polytron mixer. 12 Kg of P.M.I. Certified Rodent diet #5002M was weighed and placed into a mixer equipped with a liquid tube and high-speed liquid addition blending bar. The two solutions of test materials were added to the blender via the liquid feed tube while the diet was mixing. Each beaker was rinsed with an additional 100 ml of distilled water. After 5 minutes of mixing the blender was opened and the diet was blended for an additional 15 minutes.

Details on mating procedure:

- M/F ratio per cage: one females rat with one male from the same treatment group
- Length of cohabitation: 21 days
- Proof of pregnancy: a female was presumed pregnant if a sperm plug was detected in her vagina, or sperm plugs were found on the absorbent paper liner beneath the cage. If fewer than 3 sperm plugs were observed on the dropsheet, a vaginal lavage sample was obtained and examined microscopically.
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples from the firts diet preparation were collected and submitted for analysis to determine uniformity of mixing and to confirm the intended dietary concentrations. These samples were collected from the top, middle, and bottom of one batch at each dietary concentrations. Samples from week 5 were analyzed for proximity to target concentrattions as well as for stability of Primid XL-522 in the feed following 7 and 14 days storage at room temperature. Additional samples from preparations for weeks 11 and 21 were analyzede for proximity to target concentrations. Samples were submitted frozen and packed in dry ice to Huntingdon Research Centre LTD., Cambs.,England for analysis.
The samples were analysed with the HPLC anlysis . The results from analysis indicate that the test diets analysed during the toxicity study were accurately formulated. The results also confirm that the formulation that were homogeneous and stable for a period representing the time from preparation to completation of feeding .

Duration of treatment / exposure:

Animals were exposed to Primid XL-552 beginning at approximately six weeks of age. Animals were mated after eleven weeks of exposure; treatment continued throughout gestation, lactation and until terminal necropsy , for a total of 21 week.

Frequency of treatment:

Animals were fed ad libitum with P.M.I. Certified Rodent Diet #5002M.

Details on study schedule:

Weeks of Study Dates Study Events
-1 January 11-17, 1994 Parenteral pre-treatment
Day 0 January 18, 1994 Parenteral treatment began
0-10 January 18 April 05, 1994 Pre-mating
11-13 April 05-26, 1994 Mating
11-16 April 06-May 19, 1994 Gestation
14-19 April 27-June 04, 1994 Lactation
21 June 14-17, 1994 Parenteral Necropsy

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1000 ppm
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
4500 ppm
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
20.000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

30 animals per sex per dose

Control animals:

yes, plain diet

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Cage-side checks to detect dead or moribund animals were made twice daily throughout the in-life phase of the study. Dropsheets were also examined for abnormalities of the feces and/or urin curing the cage-side checks. An exception was made on holidays and weekends, when cage-side checks were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
All animals were examined weekly for signs of ill health, reaction to treatment, and/or abnormal behavior or appearance.

BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Male and female body weights and feed consumption were recorded weekly until cohabitation. Feed consumption was not measured during cohabitation because of the inability to determine consumption on an individual basis. Body weight and feed consumption of females were measured during gestation on Days 0,7, 14 and 21, and during lactation on Days 0, 4, 7, 14 and 21. Body weights for females that did not mate, and all males, were determined weekly until terminal necropsy. Compound intake was calculated weekly from feed consumption and body weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
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. Offsprings 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):

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/body weight) followed by exsanguination. The following tissues were collected from each animal and preserved in 10% neutral buffered formalin for histopathological examination:
Males: epidydimes, 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.

HISTOPATHOLOGY
Microscopic examination was performed on all tissues collected from control and high doser 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):

Pups found dead, and pups culled on day 4 PP were grossly examined for abnormalities of the abdominal and thoracic cavities. all remaining offsprings were euthanized by CO2 axphyxiation at the end of the weaning period ( Day 21 PP) and similarly examined for gross abnormalities.

Statistics:

The litter was used as the experimental unit for the purpose of statistical evaluation. The level of significance selected was p<0.05.
The following statistical analyses were performed:
Parameter Statistical method

Incidence of pregnancy Fisher's Exact Test
Maternal death -
Litters with stillborn -
Parental body weight Analysis of Variance (ANOVA)
Parental feed consumption -
Offspring body weight -
Lenght of gestation -
Live fetuses/litter Mann-Whitney U Test
Viability Index -
Lactation Index -
Sex ratio -
reproductive Indices Fisher's Exact Test or Mann-Whitney U Test

Reproductive indices:

Number of males that mated Number of females mated
Male Mating Index (%)=--------------------------------- x 100 Females Mating Index (%)=--------------------------------- x 100 Number of males used for mating Number of females used for mating Number of sires Number of pregnant females
Male fertility Index (%)=--------------------------------- x 100 Female fertility Index (%)=--------------------------------- x 100
Number of males mated Number of females that mated
Number of females producing litters with at least one live pup
Gestation Index (%) =------------------------------------------------------------- x 100
Number of pregnant females

Offspring viability indices:

Number of pups/litter alive on Day 4 PP
Viability Index (%) = --------------------------------------- x 100
Number of pups/litter born alive

Number of pups/litter alive at weaning (Day 21 PP)
Lactation Index (%) = ----------------------------------------------- x 100
Number of pups/litter alive after culling (Day 4 PP)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no treatment-related effects on mean body weights in male animals at doses up to and including 20.000 ppm . No treatment-related effect on body weight was observed in female anim

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no treatment-related effects on mean body weights in male animals at doses up to and including 20.000 ppm . No treatment-related effect on body weight was observed in female anim

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS) There was no treatment-related mortality in animals either sex at doses up to and including 20.000 ppm. No treatment-related clinical signs of toxicity were observed 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. 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 30 animals, only during the premating period. In most of these animals, these signs did not develop and persist until 6-7 weeks of treatment. 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.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS) a statistically significant decrease in mean body weight was observed in males exposed to 4500 ppm Primid XL-552 during weeks 6-12 of treatment. Due to the lack of dose-response, this finding was not judged to be treatment-related. Females in the 1000 ppm dose group exhibited consistently higher body weights than the control group throughout gestation and lactation, which was statistically significant at day 14 of lactation. This finding was judged to be incidental.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS) The mean compound intake for all adult animals was calculated from feed consumption and body weight data as follows:
(Dietary concentration ppm x Feed Consumption g/day)
Compound intake (mg/kg/day) =----------------------------------------------------
(Body weigth g at the beginning of the week)


GROSS PATHOLOGY /HISTOPATHOLOGY (PARENTAL ANIMALS) The only possible treatment-related gross finding was perineal staining with feces in 7 of 30 high dose male rats. These observations suggest soft feces and /or failure to groom their perineal areas . However, gross examination of the entire gastrointestinal tract did not indicate the presence of lesions that could be related to perineal staining. There were no treatment-related microscopic changes in tissues examined from male and female parental rats administered 20.000 ppmPrimid XL-552 in their diet.
In order to maximize the usefulness of the microscopic examination of the female reproductive tract, the morphology of the vagina and uterus during the estrus cycle was characterized .The uterine and vaginal morphologic changes as pertains to the estrus cycle occured with similar frequency in both high dose treated and control females.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

> 20 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive toxicity was not observed at doses up to and including 20000 ppm.

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:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Compound intake results:

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 ± 81.68
20.000	M	1280.4 ± 100.58
	F-P	1726.7 ± 105.89
	F-G	1380.5 ± 14.71
	F-L	2735.2 ± 408.87

Applicant's summary and conclusion

Conclusions:

Primid XL-552 , when administered in the diet has no observed effect level (NOEL) for general toxicity in parental animals of 4500 ppm. At 20.000 ppm, indications of toxicity were limited to soft and/or irregular feces in both sexes. Due to the delayed onset, the toxicological significance and relationship to treatment of these signs were considered equivocal. Reproductive toxicity was not observed at dose up to and including 20.000 ppm.

Executive summary:

This study was performed to investigate the effects of Primid XL-552 on growth, development and reproductive performance in male and female rats through one generation. Animals were exposed to Primid XL-552 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 throughtout gestation, lactation and until terminal necropsy. Body weight, feed consumption, and clinical signs were monitored in parental animals throughout the study. Survival and growth of offsprings were monitored throughout lactation. Offsprings that were euthanized (culled on day 4 or terminally sacrificed at day 21) and pups found dead were grossly examined. Parental animals were euthanized and necropsied after weaning of the offspring. Reproductive organs, liver, kidneys, and pituitary were examined microscopically in all control and high dose (20.000 ppm) parental animals. Since no treatment-related effects were observed, tissues were not examined at lower dose levels.

Primid XL-552 , when administered in the diet has no observed effect level (NOEL) for general toxicity in parental animals of 4500 ppm. At 20.000 ppm, indications of toxicity were limited to soft and/or irregular feces in both sexes. Due to the delayed onset, the toxicological significance and relationship to treatment of these signs were considered equivocal. Reproductive toxicity was not observed at dose up to and including 20.000 ppm.