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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The GLP-study was performed according to an internationally accepted guideline (OECD 416). All study parameters are well documented and are based on the specific guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Those animals passing the initial shipment exam were individually housed (except during the mating phase and as noted below for the F, pups) in suspended stainless steel wire-mesh cages with deotized cage board in the bedding tray. Polycarbonate cages with corn cob bedding were used to house individual dams and their litters, as well as the offspring after weaning until they were moved to individual housing.
The animals were housed in a temperature-, humidity-, and light-controlled, AAALAC-accredited facility (room temperature 18 to 26°C, relative humidity 30 to 70%, daily photo period of 12 hr of fluorescent light [6:00 a.m. to 6:00 p.m.] alternating with 12 hr of darkness) and a minimum daily average of 10 air changes per hourc.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
ethanol
Details on exposure:
The test compound was dissolved in ethanol before mixing in the diet. The control diet was prepared the same way, excluding only the test substance. Treated diet was mixed at room temperature; aliquots of the test substance were taken from the original test batch and transferred to the mixing area. A sample of each batch of feed mixed was taken and retained in the freezer until the study was complete and the analytical data deemed satisfactory. Replacement admixtures for each treatment group, for each week (or at greater intervals depending on freezer stability) were stored at freezer conditions until presented to the animals the following week (or weeks).
All animals were 9.5 weeks of age when exposure to the test substance was initiated (6/18/03) and were exposed to the treated feed seven days/week throughout the entire study. To initiate the study, 120 female and 120 male rats were assigned to one of four treatment groups (30 rats/sex/group): 0, 250, 500, or 3,000 ppm 1,2,4-triazole in the diet (based on the percent active ingredient).
Dietary levels (250, 500, and 3,000 ppm) were selected, based principally upon the toxicological profile that emerged in young-adult rats over the course of a 13-week dietary study conducted with the test chemical at dosages of 100, 500, and 2,500 ppm 1,2,4-triazole (Bomhard etal., 1979).

Details on mating procedure:
Mating was accomplished by co-housing one female with one male for up to 14 consecutive days. Approximately four to six animals from each dose group were cohoused daily beginning on the first day of the mating phase and continuing until all animals were cohoused. During the mating phase, vaginal smears were collected each morning and examined for the presence of sperm and/or an internal vaginal plug. Females that were determined to be inseminated (designated Day 0 gestation) were then placed in polycarbonate nesting cages. To evaluate females that may have been inseminated (without exhibiting sperm in the vaginal smear or an internal vaginal plug), all remaining females were placed in polycarbonate nesting cages following the mating period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of 1,2,4-triazole in the various test diets was determined using liquid chromatography (LC-MS/MS ).
Duration of treatment / exposure:
continously daily
Frequency of treatment:
continously daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 250, 500 and 3000 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
30/sex/dietary level
Control animals:
yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:twice daily during the workweek, once daily on weekends and holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once each week

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was measured weekly for males and females during the premating periode. During the mating period and until sacrifice, body weights for males and females were recorded once/week. Dams were weighed during gestation on days 0, 6, 13, and 20 and during lactation on days 0, 4, 7, 14, and 21.


FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:



OTHER:
Oestrous cyclicity (parental animals):
The estrous cycle was determined by examining daily vaginal smears over a three-week period prior to mating of the P- and F1-generation females. The vaginal smear was taken each morning at approximately the same time. Additionally, the estrous cycle stage was determined for all females just prior to termination.
Sperm parameters (parental animals):
For all P- and F,-generation males at termination (except for perfused F1-males), sperm was collected from one testis and one epididymis for enumeration of homogenization-resistant spermatids and cauda epididymal sperm reserves, respectively. In addition, an evaluation of the morphology and motility was performed on sperm sampled from the distal portion (closest to the urethra) of the vas deferens. Sperm motility and counts were conducted using the Integrated Visual Operating System (IVOS, Hamilton-Thorne Research, 1998). Morphology and counts were conducted for all levels for the 1st generation, as well as the control and high dietary group (500 ppm) for the 2nd generation.
Litter observations:
The numbers of live and stillborn pups (both F1- and F2-generations) were recorded for each litter. As soon as possible after parturition was judged complete, each pup was examined, sexed, weighed and individually identified by tattoo of the paws. Dead pups were necropsied and the lungs "floated" to determine whether the pup died after delivery or was stillborn. Additionally, the anogenital distance was measured on lactation Day 0 for all F2-pups.
F,- and F2-pups were observed daily for clinical signs (cageside, as described for adults) from birth until all pups were weaned, and weekly thereafter until the start of the premating phase (F,- and F2-pups)g. In the event a possible clinical sign was observed during the cageside evaluation, pups were removed from the cage, as needed, for a more detailed assessment. Detailed clinical observationsg, and a physical examination were performed on days the pups were weighed.
Pups were sexed and their body weights recorded as soon as possible following parturition (Day 0 of lactation). Pup body weights were also recorded on lactation Days 4, 7, 14, and 21, and when vaginal opening and preputial separation were evident.
F,- andF2-pups were observed daily for vaginal opening (beginning on Day 29) and preputial separation (beginning on Day 38)g. Additionally, anogenital distance was measured for all F2-pups on lactation Day 0.

Postmortem examinations (parental animals):
Females:
Following the weaning of their respective litters on lactation Day 21, each dam (both P-and F,- generations) was sacrificed by carbon dioxide asphyxiation and a gross external examination was performed. Terminal body weight was measured and the abdomen and thoracic cavities were opened, a gross internal examination was performed, the uterus was excised and the number of implantation sites, if any, was counted.

Females that were sperm positive and/or had an internal vaginal plug but did not deliver were sacrificed after gestation Day 24 by carbon dioxide asphyxiation. Females that were never observed to be inseminated and/or with an internal vaginal plug and that did not deliver by 24 days after the completion of the mating phase, were sacrificed. A gross necropsy was performed on these animals, as described above, including measurements of term body weight, estrous cyclicity, organ weights, as well as organ preservation. These females were also examined for the patency of the cervical/uterine os (via flushing of the uterine horns with 10% buffered formalin). The following tissues were also collected and preserved in 10% buffered formalin: vagina, physical identifier and grosslesions. The ovary was also collected and fixed in Bouin's fixative. All paired organs were weighed individually.

Males:
The testes, epididymides (total weight for both, and cauda weight for the side not used for sperm analysis), seminal vesicles (with coagulating glands and their fluids), prostate, brain, pituitary, thymus, liver, kidneys, adrenal glands, thyroids and spleen were removed, weighed, and fixed in 10% buffered formalinh. The following tissues were also collected and preserved in 10% buffered formalin: physical identifier and gross lesions. One testicle (the side not used for sperm analysis) was collected and fixed in Bouin's fixative. All paired organs were weighed individually.

The following tissues from adult animals (P- and F1-generations) were examined microscopically: brain, spleen, kidneys, cervix, epididymis (caput, corpus, and cauda), gross lesions, adrenal glands, liver, ovaries, pituitary, prostate, testis, seminal vesicles/coagulating gland, uterus, oviducts, and vagina.
Postmortem examinations (offspring):
Culled pups were sacrificed by intracranial injection of 0.01-0.05cc Fatal Plus (Vortech Pharmaceuticals, Dearborn, MI). Grossly abnormal pups underwent a gross internal and external examination, and all culled pups were discarded.
The F1- and F2-pups not culled on lactation Day 4 were maintained with the dam until weaning at 21-days of age. At 21-days of age, sufficient numbers of F,-pups/sex/litter were maintained to produce the next generation. There were too few pups at 3,000 ppm (one pup each from two dams) to foster a second generation; therefore, all high-dose females and their F,-pups were sacrificed and processed before weaning. F,-pups not selected to become parents for the next generation and all F2-pups not selected for sexual maturation were sacrificed and examined macroscopically for any structural abnormalities or pathological changes, particularly as they may have related to the organs of the reproductive system.
Pups found dead or terminated in a moribund condition were examined for possible defects and/or cause of death.
For pups that were sacrificed on lactation Day 21, the brain, spleen, thymus, and uterus were weighedh. One pup/sex/litter from each generation had the following reproductive tissues collected for microscopic examination: uterus, ovaries, vagina, cervix, oviduct (fallopian tube), testes, epididymides, prostate, coagulating gland, and seminal vesicles. The computer (SAS) printout obtained for four-day-old culls was used to select these pups. Tissues that exhibited evidence of a potential compound-related effect and target organs (when known) were preserved for possible microscopic examination.

The following tissues from one pup/sex/litter (F1- and F2-generations) were examined microscopically: uterus, ovaries, vagina, cervix, oviduct (fallopian tube), testes, epididymides, prostate, coagulating gland, and seminal vesicles.

On PND 21, the whole brain (with olfactory bulbs) was collected from randomly-selected pups (10/sex/dietary level, representing 20 litters/dietary level) for micropathologic examination . Each of these brains was divided into eight coronal levels for microscopic examination.

The brains from animals that received the high dose were examined relative to those from controls. If treatment-related neuropathology was evident, samples from the next lower dose group were examined in sequence until the lesion was no longer evident. Any region where treatment-related neuropathology was observed underwent the following semi-quantitative analysis: The frequency of each type of lesion was determined, with the severity of each lesion graded. The code was then broken to reveal the dose group assignments and the data were evaluated for dose-effect relationships.


Reproductive indices:
The 3,000 ppm dietary level was associated with a marked reduction in fertility in the first generation, with only two high-dose females delivering viable offspring (one each) and only three implantation sites (compared to 265 for controls). Notably, the 28 high-dose females that did not deliver any pups were not pregnant (no implantation sites). All high-dose (3,000 ppm) P-generation females and pups were sacrificed before weaning, since there were too few pups to provide a second generation. At the 250 and 500 ppm dietary levels, there were no compound-related effects on any parameter (e.g., mating, fertility, or gestation indices, days to insemination, gestation length, or the median number of implants) in either generation.
Offspring viability indices:
There were no test substance-related clinical observations or effects on body weight, nor were there effects on the age for onset of vaginal patency or preputial separation in either of the 250- or 500-ppm dose groups.

The statistical differences associated with the parameters listed below are considered to be attributed to an incidental difference in the time of delivery (i.e., post-conception age) for both the 250- and 500-ppm dietary groups, relative to concurrent controls, rather than exposure to the test substance.

The combined (male and female) F2-pup body weights for the 250- and 500-ppm dietary groups were significantly different from control and are considered to be due to the incidental difference in the time of delivery, as discussed above, and not exposure to the test substance. As seen in Text Table 7 (below), body weights for the 250- and 500-ppm dose groups were well within the range for historical control values while the concurrent control values (with the exception of Day 14) were above the range for historical values. The heavier pups observed in the concurrent control group (due to more control dams delivering on gestation Day 23 and fewer on day 21) contributed to the statistical significance that was observed for both treated groups. Furthermore, body weights and weight gains for both the treated groups (as well as the concurrent control) approached the high end or exceeded (rather than the low end or below) the range of historical values.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30.9 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: body weight, brain weight, cerebellar degeneration/necrosis
Dose descriptor:
NOAEL
Effect level:
36.2 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: body weight, brain weight, cerebellar degeneration/necrosis, corpora lutea, uterine dilatation
Dose descriptor:
NOAEL
Effect level:
34.4 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fertility index; number of implantation sites
Dose descriptor:
NOAEL
Effect level:
35.8 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental

Results: P1 (second parental generation)

Effect levels (P1)

Key result
Dose descriptor:
LOAEL
Effect level:
16 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decrease body weight and body weighty gain during premating in F1 parent generation
Remarks on result:
other: decrease body weight and body weighty gain during premating in F1 parent generation

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
37.5 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
16 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Slight decrease in body weight in F1 males only

Results: F2 generation

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
35.8 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effet at the high dose -level

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
231.7 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
Conclusion from study report:
1,2,4-triazole produced considerable evidence of toxicity in P-generation animals at a dietary level of 3,000 ppm (188.6 and 217.9 mg/kg/day for males and females, respectively), including reduced fertility and neuropathology. By comparison, the only evidence of toxicity at lower dietary levels of 250 and 500 ppm was slightly reduced body weight in Fr-generation males. Based on these results, the reproductive (fertility) NOAEL is 34.4 mg/kg/day and the LOAEL is 231.7 mg/kg/day, based on findings in P-generation animals. The developmental NOAEL is 35.8 mg/kg/day and the LOAEL is greater than 35.8 mg/kg/day. No definitive parental NOAEL was established, a slight decrease in body weight in the F1-generation males was observed at 16.0 mg/kg/day (250 ppm), but these findings were minor, seen only in males of one generation and not reported from any other avaialble study at this level. For females, the parental NOAEL was demonstrated to be 36.2 mg/kg/day (500-ppm). These results, including an extensive investigation of brain morphology, provided no evidence of developmental neurotoxicity at a dietary level of 500-ppm.
Executive summary:

1,2,4-triazole produced considerable evidence of toxicity in P-generation animals at a dietary level of 3,000 ppm (188.6 and 217.9 mg/kg/day for males and females, respectively), including reduced fertility and neuropathology. By comparison, the only evidence of toxicity at lower dietary levels of 250 and 500 ppm was slightly reduced body weight in Fr-generation males. Based on these results, the reproductive (fertility) NOAEL is 34.4 mg/kg/day and the LOAEL is 231.7 mg/kg/day, based on findings in P-generation animals. The developmental NOAEL is 35.8 mg/kg/day and the LOAEL is greater than 35.8 mg/kg/day. No definitive parental NOAEL was established, a slight decrease in body weight in the F1-generation males was observed at 16.0 mg/kg/day (250 ppm), but these findings were minor, seen only in males of one generation and not reported from any other available study at this level. For females, the parental NOAEL was demonstrated to be 36.2 mg/kg/day (500-ppm). These results, including an extensive investigation of brain morphology, provided no evidence of developmental neurotoxicity at a dietary level of 500-ppm.