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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - June 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

sub-chronic oral toxicity was assessed embedded in an OECD 415 One-generation reproduction toxicity study

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

Constituent 2

Reference substance name:

188132-16-3

Cas Number:

188132-16-3

IUPAC Name:

188132-16-3

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

other: Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
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., KingstonFacility, 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 acclimation 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 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) suspended above absorbent paper liners that 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 (21 in. x 11.5 in. x 8 in.; 53.3 cm x 29.2 cm x 20.3 cm) containing Alpha-Dri bedding (Shepherd Specialty Paper, Kalamazoo, MI). Filtered tap water was available ad libitum via an automatic watering system. Females during gestation and lactation received water ad libitum via a water bottle. Animals were fed ad libitum with P.M.l. Certified Rodent Diet #5002M. The study rooms were environmentally controlled with controls set to maintain a temperature of approximately 73 °F (22.8 °C) with a relative humidity range of 40 - 60%. Temperature and relative humidity were monitored 24 hours a day. During the study the average daily temperature ranged from 69 – 75 °F (21 – 24 °C), and average daily humidity ranged from 40 - 53%, with the exception of a single day when average humidity fell to 35%.
Throughout the study, temperature and humidity remained in general compliance with acceptable ranges defined in the “Guide for the Care and Use of Laboratory Animals" NIH Publication No. 86-23, Revised 1985. Excursions in humidity levels beyond these ranges were minimal and did not affect the integrity of the study. An automatically controlled 12 hour light/dark cycle was maintained throughout the study.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Exposure of parental animals was performed through fee; details see below.

Vehicle:

water

Remarks:

water as solvent for test substance was used to prepare feed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

DIET PREPARATION AND SAMPLE ANALYSIS

Group Substance concentration (ppm) Amount required
for 12 kg of feed (g)* Final volume
in distilled water (ml)
1 0 0.0 480
2 1’000 12.0 480
3 4’500 54.0 480
4 20’000 240.0 480
* Dietary preparations were based on total product.
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.l Certified Flodent Diet #SOOZM 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 tube while the diet was mixing.
Each beaker was rinsed with an additional 100 ml of distilled water. which was added via the liquid feed tube to the mixing diet. After 5 minutes of mixing. the blender was opened and the sides were scraped. The diet was blended for an additional 15 minutes. 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.
Samples from the first 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 concentration. Samples from Week 5 were analyzed for proximity to target concentrations as well as for stability of Primid XL-552 In the feed following 7 and 14 days storage at room temperature. Additional samples from preparations for weeks 11 and 21 were analyzed for proximity to target concentrations. Samples were submitted frozen and packed in dry ice to Huntingdon Research Centre LTD., Cambs., England for analysis.

Duration of treatment / exposure:

21 weeks until necropsy for parental animals

Frequency of treatment:

continuous via feed

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

1 000 ppm

Remarks:

Low dose group equivalent to 62.6 mg/kg bw/d for males and 85.9, 69.5 and 122.6 mg/kg bw/d for females during pre-mating, gestation and lactation, respectively.

Doses / concentrations 3

Dose / conc.:

4 500 ppm

Remarks:

Mid dose group equivalent to 284.2 mg/kg bw/d for males and 387.5, 294.1 and 562.6 mg/kg bw/d for females during pre-mating, gestation and lactation, respectively.

Doses / concentrations 4

Dose / conc.:

20 000 ppm

Remarks:

High dose group equivalent to 1280.4 mg/kg bw/d for males and 1726.7, 1380.5 and 2735.2 mg/kg bw/d for females during pre-mating, gestation and lactation, respectively.

No. of animals per sex per dose:

30 males and 30 females per dose group

Control animals:

yes, plain diet

Details on study design:

PRE-MATING
Males and females, 6 weeks of age were acclimatised for one week and then subject to a pre-mating period of 11 weeks, during which exposure to the test substance through diet was performed; drinking water was provided ad libitum.
MATING and GESTATION
Female rats were cohabited individually with an assigned male from the same treatment group in suspended, wire-mesh. stainless steel cages (13.5 in. x 7 in. x 7 in.; 34.3 cm x 17.8 cm x 17.3 cm). Females were observed daily for positive signs of mating. 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 for the presence of sperm to verify mating. The day that evidence of mating was found was designated Day 0 of gestation (Day 0 G).
Females without a confirmed copulation date after 10 days of cohabitation were placed with a different male from the same treatment group. A male was used that had already copulated successfully. 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.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

IN-LIFE OBSERVATIONS: PARENTAL ANIMALS
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 urine during the cage-side checks. An exception was made on holidays and weekends, when cage-side checks were made once daily. All animals were examined weekly for signs of ill health, reaction to treatment, and/or abnormal behaviour or appearance.
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.

LACTATION AND IN-LIFE OBSERVATIONS: OFFSPRING
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 behaviour or appearance on Days 0, 4, 7, 14 and 21 PP.
Litters were culled randomly by computer selection to B pups (4 per sex) on Day 4 PP. If a litter did not have 4 offspring per sex, then the litter was culled to 8 by computerized random selection from the remaining offspring. Litters with 8 offspring or less were not culled. Pups selected for culling were euthanized via IP injection of Nembutal®.

REPRODUCTIVE INDICES
The following indices of reproductive performance were calculated for each treatment group or individual animal, as appropriate:
Male Mating lndex (%) = 100 * Number of males that mated/Number of males used for mating
Female Mating Index (%)= 100 * Number of females mated/Number of females used for mating
Male Fertility lndex (%) = 100 * Number of sires/Number of males mated
Female Fertility index (%):100 * Number of pregnant females/Number of females that mated
Gestation Index (%) = 100 * Number of females producing litters with at least one live pup/Number of pregnant females
Viability Index (%) = 100 * Number of pups/litter alive on day 4 PP/Number of pups/litter born alive
Lactation Index 0%) = 100 * Number of pups/litter alive at weaning (Day 21 PP)/Number of pups/litter alive after culling (Day 4 PP)

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 axphyxiation at the end of the weaning period (Day 21 PP) and similarly examined for gross abnormalities.
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. 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 sex: 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.

Sacrifice and pathology:

HISTOPATHOLOGY
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.

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. The following statistical analyses were performed:
Incidence of pregnancy, Maternal death and Litters with stillborn --> Fisher‘s Exact Test (2)
Parental body weight, Parental feed consumption, Offspring body weight and Length of gestation --> Analysis of Variance (ANOVA)
Live fetuses/litter, Viability Index, Lactation Index and Sex ratio --> Mann-Whitney U Test
Reproductive lndices --> Fisher's Exact Test or Mann-Whitney U Test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

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 faces 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 30 animals, only during the pre-mating 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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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 (Animal No. 93-03167) was euthanized for humans reasons during week 12 due to weight loss and discomfort resulting from malocclusion.One female in the 1000 ppm treatment group (Animal No. 93-03304) was found dead following a prolonged delivery. Gross necropsy revealed the presence of two pups apparently lodged in the cervix. Microscopic examination revealed pyometra (accumulation of pus in the uterus) was the cause of death in this animal. One female in the 1’000 ppm group (Animal No. 93-03245) was sacrificed moribund during lactation following weight loss, clinical signs of ill health, and the death of her entire litter. The cause of this condition could not be determined. Another female in the 1’000 ppm treatment group (Animal No. 93-03230) was sacrificed for humane reasons on Day 29 of gestation (this animal failed to deliver a litter after showing positive signs of mating) when she began dragging her hind legs. Lymphosarcoma was the cause of this condition. These findings were not considered treatment-related due to their spurious occurrences, and lack of a dose-response.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

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 4’500 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.
No treatment-related effect on body weight was observed in female animals prior to mating, during gestation, or during lactation at doses up to and including 20’000 ppm. Females in the 1’000 ppm dose groups 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.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

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 1’000 ppm during week 19, and in the 4’500 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.

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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 ppm Primid 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 occurred with similar frequency in both high dose treated and control females. In summary, 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.

Other effects:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on the male mating index or male fertility index at any dose level.
There were no treatment-related effects on the female mating index, fertility index or gestation index. Only a single female, in the 1’000 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 Primid XL-552 exposure. There were no litters that were entirely stillborn at any dose level. In only two litters (both in the 4’500 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.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

20 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: soft and/or irregular feces considered non-adverse as equivocal

2

Dose descriptor:

NOEL

Effect level:

4 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Soft and/or irregular feces in both sexes, considered equivocal due to their delayed onset

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

The mean compound intake for all adult animals was calculated from feed consumption and body weight data as follows:

Compound Intake (mg/kg/day) = dietary concentration (ppm) * feed consumption (g/day) / body weight (g) at the beginning of the week

Results were as follows:

 

Dietary Concentration (ppm)	Sex	Compound Intake (mg/kg/day) ±SE
1’000	M	62.6 ±4.90
	F-P	85.9 ±5.11
	F-G	69.5 ±1.01
	F-L	122.6 ±19.11
4’500	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	1728.7 ±105.39
	F-G	1380.5 ±14.71
	F-L	2735.2 ±408.87

M: males throughout treatment (excludes the mating period);

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.

DIET ANALYSIS

Dietary samples containing Primid XL-552 were analysed 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. Primid XL-552 was stable in the diet for at least 14 days when stored at room temperature (approximately 68 °F; 20 °C).


3. Primid XL-552 concentrations were within ±10% of nominal target concentrations for all levels, with one exception. A single diet preparation of 1’000 ppm was 35.1% 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.

Table: Parental body weight (g) development

Week			-1			0		1		2		3		4		5		6		7		8		9		10		11		12		13		14		15		16		17		18		19	20
Male 0 ppm
mean			179.3			246.6		312.6		373.2		416.5		459.6		493.0		518.1		543.2		566.5		587.6		605.5		619.0		615.8		623.5		642.1		652.5		667.1		678.7		690.4		697.8	713.9
SE			2.79			3.27		3.80		4.21		4.82		5.99		6.92		7.45		8.12		8.72		9.16		9.73		10.47		10.16		10.03		10.35		10.74		10.65		11.61		11.73		12.17	12.69
#			30			30		30		30		30		30		3C		30		30		30		30		30		30		30		30		30		30		30		30		30		30	30
Male 1’000 ppm
mean		179.1			242.6			306.9		365.6		408.5		445.6		474.1		498.0		520.3		540.2		559.4		574.8		588.3		584.1		591.3		609.3		617.4		631.0		641.2		651.5		656.8	663.2
SE		2.79			3.86			4.72		5.97		7.09		9.12		10.35		11.18		11.83		12.32		13.01		13.46		13.63		13.99		13.89		14.59		14.80		14.94		15.05		14.78		15.75	16.29
#		30			30			30		30		30		30		30		30		30		30		30		30		30		30		30		30		30		30		35		30		30	30
Male 4’500 ppm
mean	178.9			239.9			303.6		360.0		400.1		437.1		467.0		486.6		507.8		527.6		548.0		558.0		575.0		575.3		585.9		602.9		611.3		627.2		636.1		649.5		655.2		664.8
SE	2.72			3.06			3.68		4.28		4.82		5.62		6.39		7.39		8.27		8.96		9.54		10.84		10.47		10.37		10.15		10.79		11.29		12.19		12.41		12.39		13.04		13.69
#	30			30			30		30		30		30		30		30		30		30		30		30		30		30		29		29		29		29		25		29		29		29
Male 20’000 ppm
mean	170.0			243.1			310.0		367.2		409.7		446.6		470.7		502.0		524.2		546.0		565.6		501.1		595.0		589.3		596.0		611.2		617.7		611.2		640.9		652.2		660.9		668.6
SE	2.73			3.74			5.10		6.10		7.12		0.24		9.30		9.06		10.50		11.15		11.97		13.25		13.14		12.17		12.53		13.15		13.84		14.51		15.60		15.40		15.98		16.09
#	30			30			30		30		30		30		30		30		30		30		30		30		30		30		30		30		30		30		30		30		30		30
Female 0 ppm
mean	139.8			172.6			200.2		227.2		247.5		165.8		277.7		290.2		300.0		309.0		319.1		325.7		333.9		326.5		358.5		393.5		484.1		370.0		378.1		381.3		373.3		357.5
SE	3.01			3.47			4.06		4.64		5.25		5.70		6.13		6.90		7.55		7.94		8.50		8.90		9.27		8.09		8.84		9.51		11.50		10.84		9.54		9.19		7.81		6.94
#	30			30			30		30		30		30		30		30		30		30		30		30		30		21		22		22		22		25		25		25		25		25
Female 1’000 ppm
mean	139.7			172.0			202.1		229.3		251.6		270.5		284.0		296.1		306.9		316.7		325.7		334.8		342.4		341.4		374.7		409.4		504.7		294.4		394.0		396.6		400.0		372.7
SE	2.95			3.26			4.04		4.18		5.00		5.33		5.64		5.93		6.44		6.76		7.12		7.52		7.56		8.93		9.20		10.10		10.67		8.66		8.12		7.57		7.23		7.79
N	30			30			30		30		30		30		30		30		30		30		30		30		30		20		20		20		19		23		23		23		23		23
Female 4’500 ppm
mean	139.9			172.4			203.6		233.0		253.8		273.1		284.8		297.9		309.0		318.3		327.2		334.7		342.9		343.7		371.8		403.6		477.9		370.4		373.1		383.6		385.0		361.0
SE	2.93			3.65			4.35		4.95		5.38		5.80		5.93		6.66		6.94		7.18		7.46		7.82		8.07		11.01		10.56		11.15		14.62		11.46		10.81		8.23		7.87		7.78
#	30			30			30		30		30		30		30		30		30		30		30		30		30		19		19		19		19		24		24		23		23		23
Female 20’000 ppm
mean	140.0			172.3			201.9		228.8		249.5		269.1		282.6		289.9		302.2		311.3		320.8		324.7		331.3		319.6		350.0		379.7		471.7		358.1		360.5		367.3		372.3		349.9
SE	3.04			2.99			3.67		4.17		4.51		4.88		5.48		5.37		5.58		5.91		6.48		7.31		7.24		6.71		6.54		7.07		9.15		7.79		7.80		6.39		5.24		4.68
#	30			30			30		30		30		30		30		30		30		30		30		30		30		20		20		20		19		22		22		22		22		22

 

Average pup body weights in g

	0 ppm					1000 ppm					4500 ppm					20’000 ppm
	LD0	LD4	LD7	LD14	LD21	LD0	LD4	LD7	LD14	LD21	LD0	LD4	LD7	LD14	LD21	LD0	LD4	LD7	LD14	LD21
mean	6.5	11.1	18.1	36.3	54.7	6.8	11.6	18.4	37.5	55.3	6.5	11.4	18.5	37.4	57.1	6.6	10.8	17.1	35.8	54.1
SD	0.2	0.3	0.4	0.7	1.1	0.2	0.6	0.8	1.1	1.4	0.2	0.4	0.6	0.8	1.5	0.1	0.4	0.7	0.8	1.0
# females delivered	25	25	25	25	25	23	23	23	23	23	23	22	22	22	22	22	22	22	22	22

 

Pathology report

Six week old male and female CRL:CD®BR rats were fed diets containing 0, 1000, 4500 or 20000 ppm Primid XL-552 for approximately eleven weeks prior to mating and throughout the mating, gestation and lactation periods and until terminal necropsy. The number of live, dead or stillborn offspring was determined. All parental (P1) males and females were necropsied and selected tissues were examined microscopically. This report characterizes the gross and microscopic findings in P1 animals.

Material and Methods: All organs, tissues and body cavities were examined from P1 animals scheduled for necropsy and those found dead or sacrificed moribund. All animals found dead were necropsied immediately or refrigerated until the necropsy could be performed. Parental rats were euthanized by intraperitoneal injection of Nembutal® followed by exsanguination. The following tissues were collected: liver, kidneys, pituitary gland, ovaries, uterus, cervix, vagina, testes, epididymides, prostate, seminal vesicles, coagulating gland, and other tissues with gross changes. The tissue samples were preserved in 10% neutral buffered formalin.

Microscopic examination was conducted on all of the above tissues from control and high dose groups of adult male and female P1 rats and from P1 rats which died or were killed moribund. Since no treatment-related microscopic findings were observed, only tissues with gross changes from the intermediate dose groups scheduled for necropsy were examined microscopically. Tissues examined were routinely processed, sectioned at approximately 5-6 microns, stained with hematoxylin and eosin and examined by light microscopy.

Results: All P1 rats except for 1 male and 3 females survived to study termination. The early death animals were from intermediate dose groups. Their conditions were not related to treatment with Primid XL-552. The male (93-03167) was killed moribund due to malocclusion. One female (93-03230) had Iymphosarcoma as the cause of her moribund condition, pyometra was the cause of death in another female (93-03304) and the cause of the moribund condition in one female (93-03245) could not be determined. 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 for 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 the tissues examined from male and female P1 rats administered 20000 ppm Primid XL-552 in their diet.

The most commonly observed microscopic finding in male rats was glomerulonephropathy, either unilateral or bilateral. It occurred with approximate equal frequency in high dose treated and control males. In this study, the early phases of this spontaneous and progressive condition were observed and included dilated and protein filled tubules, hypertrophy of tubule epithelium, peritubular fibrosis and focal interstitial inflammation. A grading scheme was devised in order to assure that a treatment-related exacerbation did not occur. A grade of slight was given if approximately 5% or less of the parenchyma from either kidney was affected. As shown in Table 3, a grade of slight occurred in all affected control and high dose animals.

Other non treatment-related microscopic changes in male rats included atrophy of spermatic elements in seminiferous tubules in a testicle along with absence of sperm in the corresponding epididymis. Tubule atrophy is a common age-associated change and can be either unilateral or bilateral (Takahashi, et al., 1992).

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 following estrus cycle data was modified from Brown and Leininger, 1992.

As can be seen in Table 3, the uterine and vaginal morphologic changes as pertains to the estrus cycle occurred with similar frequency in both high dose treated and control females.

Incidental microscopic changes in female rats included the presence of a benign endometrial polyp in a high dose female, cystic endometrial hyperplasia in a control female and pyometra in one intermediate dose level female. Endometrial stromal polyp is the most commonly diagnosed spontaneous neoplasm in the uterus of the rat (Brown and Leininger, 1992). Pyometra, or the accumulation of purulent material in the uterine lumen was the cause of death in the affected intermediate dose level female.

in summary, dietary administration of 20000 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.

Applicant's summary and conclusion

Conclusions:

Primid XL-552, when administered in the diet has a no observed effect level (NOEL) for general toxicity in parental animals of 4’500 ppm. At 20’000 ppm, indications of toxicity were limited to soft and/or irregular feces in both sexes. Due to their delayed onset, the toxicological significance and relationship to treatment of these signs were considered equivocal. Reproductive toxicity was not observed at doses up to and including 20’000 ppm.

Executive summary:

Exposure of rats to Primid XL-552 from approximately six weeks of age, into adulthood, and through production of one generation of offspring had the following results:

1’000 ppm Compound Intake: Males: 62.6 mg/kg/day, Females during Pre-mating: 65.9 mg/kg/day, gestation: 69.5 mg/kg/day and lactation: 122.6 mg/kg/day

Parental animals: No treatment-related effects

Reproductive effects: No treatment-related effects

4’500 ppm Compound Intake: Males: 264.2 mg/kg/day; Females during Pre-mating: 367.5 mg/kg/day, gestation: 294.1 mg/kg/day and lactation: 562.6 mg/kg/day

Parental animals: No treatment-related effects

Reproductive effects: No treatment-related effects

20.000 ppm Compound Intake: Males: 1260.4 mg/kg/day; Females during Pre-mating: 1726.7 mg/kg/day, gestation: 1360.5 mg/kg/day and 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