Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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.
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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.
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
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
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.
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
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
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
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
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
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
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
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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
16 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

According to the HPV dossier (US EPA 2009, 1H-1,2,4 -triazole) :

In an OECD Guideline 416 "Two-generation Reproduction Toxicity Study", TA was administered continuously via the feed to Wistar Hannover rats (30/sex/dietary level) at nominal dietary concentrations of 0, 250, 500, and 3000 ppm (Bayer, 2005; RCC, 2003; TDMG, 2006). To maintain a more constant dosage (on basis of mg/kg bw/day) throughout the entirety of the study the dietary levels were reduced for females during lactation. In addition to fulfilling standard guideline requirements, this two-generation reproduction study contained the following additional investigations: (1) in-depth examinations of brain tissue from the P- and F1-generation adults, as well as the F1- and F2-generation 21-day-old pups, which included qualitative microscopic evaluations and gross and microscopic morphometric analyses; (2) the inclusion of additional microscopic investigative work on the ovarian tissue from the P-generation rats as it related to the identification of infertility in the 3000 ppm group during the conduct of this study; (3) a more in-depth study of the corpora lutea, based on findings from the P-generation ovarian data; and (4) determination of the onset of preputial separation and vaginal patency for F2 pups. There were no effects on food consumption or clinical signs in either generation at any dietary level. Compound-related declines in body weight and body weight gain were evident in the P-generation adult males and females of the 3000-ppm dose group. A slight decrease in body weight and weight gain that was attributed to treatment was also evident in F1-generation adult males in both the 250 and 500ppm dose groups. A marked reduction in fertility was evident at the 3000 ppm dietary level of the P-generation, with only two females delivering viable offspring (one each) and only three implantation sites (compared to 265 for controls). All high-dose (3000 ppm) P-generation females and pups were sacrificed before weaning, since there were too few pups to provide a second generation. There were no test substance-related effects on the mating, gestation, or fertility indices, number of days to insemination, or gestation length at any dietary level in either F1- or F2- generation except for decreased fertility in 3000 ppm P-generation females. No effects were observed on the oestrous cycle length or the number of cycles at any dose in either generation, including the 3000 ppm group of the P-generation. Other than the reduced fertility evident in the 3000 ppm dose group, no other effects were observed on any litter parameter in any dose group in either generation. There was also no effect on any sperm parameter that was attributed to the test substance. At termination, treatment-related findings were evident only in the P-generation at the 3000 ppm dietary level and included: 1) decreased absolute brain weights in males and females; 2) increased incidence of cerebellar degeneration/necrosis in both genders; 3) statistically increased number of total Corpora lutea measured by quantitative ovarian analysis and increased ovary weights; and 4) increased incidence of uterine horn dilatation. No similar findings were evident in P-generation animals at lower dietary levels or in the offspring from either generation. Based on these results, the reproductive (fertility) NOAEL is 34.4 mg/kg bw/day (500 ppm) and the LOAEL is 231.7 mg/kg bw/day (3000 ppm), based on findings in P-generation animals. No definitive parental NOAEL was established, based on a slight decrease in body weight in the F1-generation males at 16.0 mg/kg bw/day (250 ppm). However, for females, the parental NOAEL was demonstrated to be 36.2 mg/kg bw/day (500 ppm).


Short description of key information:
In a two-generation study, TA produced 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 F1-generation males. Based on these results, the reproductive (fertility) NOAEL is 34.4 mg/kg bw/day (500 ppm) and the LOAEL is 231.7 mg/kg bw/day (3000 ppm), based on findings in P-generation animals. No definitive parental NOAEL was established, based on a slight decrease in body weight in the F1-generation males at 16.0 mg/kg bw/day (250 ppm). However, for females, the parental NOAEL was demonstrated to be 36.2 mg/kg bw/day (500 ppm).

Justification for selection of Effect on fertility via oral route:
The longest duration study available with the lowest LOAEL. A slight decreased in body weight of the F1 male generation.

Effects on developmental toxicity

Description of key information
In an OECD Guideline 414 "Teratogenicity" study with pregnant Bor: WISW (SPF Cpb) rats the maternal NOAEL was = 30 mg/kg bw/day based on retarded weight gain at 100 mg/kg bw and the developmental NOAEL was = 30 mg/kg bw/day based on an increased incidence of runts and lower fetal weights at 100 mg/kg bw/day.
In an OECD Guide-line 414 "Teratogenicity" study with pregnant New Zealand White rabbits [Hra: NZW:SPF], the maternal NOAEL was = 30 mg/kg bw bw/day based on mortality, reduction in body weight gain, and clinical symptoms at 45 mg/kg bw/day and the developmental NOAEL was = 30 mg/kg bw/day based on lower fetal weights and a slight increase in urogenital alterations for several fetuses at 45 mg/kg bw/day.
In an OECD Guide-line 414 "Teratogenicity" study with pregnant Bor: WISW (SPF Cpb) rats, the maternal NOAEL was < 100 mg/kg bw/day based on retarded weight gain at 100 mg/kg bw and the developmental NOAEL was < 100 mg/kg bw/day based on an increased incidence of runts, lower fetal and placental weights, and a higher incidence of minor skeletal deviations at 100 mg/kg bw.
In an OECD 416 “Two Generation Reproductive Toxicity” study with Wistar Hannover rats, the developmental NOAEL was > 500 ppm (equivalent to > 35.8 mg/kg bw/day) based on lack of treatment-related effects in F1 and F2 pups at 250 and 500 ppm.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The GLP-study was performed according to an internationally accepted guideline (US EPA 83-3). All study parameters are well documented and are based on the specific guideline.
Qualifier:
according to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
other: Bor: WISW (SPF Cpb)
Route of administration:
oral: gavage
Vehicle:
other: 0.5 % aqueous Cremophor-EL emulsion
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
Mating was carried out overnight: one male was placed with two females in a type III Makrolon cage. If sperm were detected in the vaginal swab on the morning after mating this counted as day zero of pregnancy.
Duration of treatment / exposure:
25 inseminated Wistar rats per group received 1,2,4-triazole on days 6 to 15 of pregnancy in daily oral doses of 0, 10, 30 or 100 mg/kg bodyweight.
Frequency of treatment:
once daily
Duration of test:
The females underwent Caesarian section on day 20 of pregnancy.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Doses of 0, 10, 30 and 100 mg/kg bodyweight were chosen for the tests. The
compound was orally administered as oral administration is well-suited and
internationally-recognised in embryotoxicological studies. Dosages were fixed
on the basis of a preliminary study using doses of 1000 and 3000 mg/kg which
had a high maternal toxicity.
The investigations were carried out in the Laboratory of Reproduction
Toxicology, the Institute of Toxicology, Agrochemicals , of the Fachbereich
Toxikologie, , Wuppertal, Friedrich-Ebert-Strasse 217 - 333 during the
period February to March 1985. They were carried out in agreement with the
OECD guidelines for good laboratory practice
Maternal examinations:
Appearance and behaviour, mortality, body weight-gain
Ovaries and uterine content:
The females underwent Caesarian section on day 20 of pregnancy. The following investigations were carried out:

a. Determination of number of nidations

b. Determination of number of foetuses/embryos that survived or died (dams without surviving foetuses were evaluated as not pregnant)

c. Determination of sex of all surviving foetuses

d. Determination of weight of each foetus and average foetal weight per litter as well as runts (for definition see page 19)

e. Determination of total and average placental weight per litter

f. Detailed examination of all foetuses for external malformations

g. Investigation of a number of foetuses for visceral malformations according to the modified WILSON technique (1,2)

h. Evisceration of remaining foetuses and evaluation of abdominal and chest organs with subsequent cleaning of the foetuses with dilute potassium hydroxide solution and staining of the bone system with alizarin red S and evaluation of the bone system (DAWSON).
Statistics:
The following statistical methods were used to test for significance:
a. Distribution-free rank sum test according to WILCOXON
(WILCOXON-MANN-WHITNEY-U-TEST), e.g. for weight-gain, number of
nidations, foetuses and resorptions.
The calculations were carried out in the Institute of Biometrics PH-DB, BAYER
AG within the framework of an evaluation programme developed for
embryotoxicitY tests.
b. Chi-squared test (correction after YATES), e.g. for number of runts
c. Chi-squared test (correction after YATES or so-called exact test
according to FISHER - depending on frequency expected) for numbers of
fertilised and pregnant animals.
These calculations were carried out with an HP 97 in case there were abnormal
differences compared with the controls.
Statistical significance has been taken as a probability of 5% or less of this
difference occurring by chance.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean weight-gain in the study groups during administration and throughout
pregnancy is given in the following table (for individual results see tables 1
to 4 in appendix):
Study group mean weight-gain of
pregnant animals in grams during
administration entire pregnancy
Control 28.2 92.9
10 mg/kg 25.4 86.6
30 mg/kg 26.8 90.0
100 mg/kg 21.8* 79.8*
* p < 0.05
Treatment with 1,2,4-triazole impaired the weight-gain of the pregnant animals
in the 100 mg/kg group.
Number of abortions:
no effects observed
Description (incidence and severity):
1,2,4-triazole produces no effect on the number of pregnancies. One animal in
group 2 was not evaluated as medication was not properly administered.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The weight-gain of the dams in the 100 mg/kg group was lower than the control animals throughout the entire pregnancy as well as during administration.The following parameters were unaffected up to a dose of 30 mg/kg
1,2,4-triazole:
number of surviving foetuses per litter
number of resorptions per litter
mean foetal and placental weight per litter
number of foetuses with slight bone changes per litter
number of runts per litter (for definition see page 19)
number of foetuses with malformations per litter
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: maternal toxicity
Remarks on result:
other: decrease maternal body weight
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The significantly lower foetal weight and the simultaneously greater number of
runts in the 100 mg/kg group can be seen in conjunction with maternal
toxicity. The lower number of male foetuses in the 10 mg/kg and 100 mg/kg
group must be coincidenta
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Foetal weight in the 100 mg/kg group was reduced and there was a higher incidence of runts.
1,2,4-triazole T5019339
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
fetal/pup body weight changes
other: decrease fetal body weight and increase number of runts
Abnormalities:
not specified
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
Conclusion from study report:
25 inseminated Wistar rats per group received 1,2,4-triazole on days 6 to 15 of pregnancy in daily oral doses of 0, 10, 30 or 100 mg/kg bodyweight.

The dams were examined with regard to bodyweight, appearance and behaviour. The foetuses obtained by Caesarian section on day 20 of pregnancy were investigated for embryotoxic effects by weighing and by observing the external and internal morphological changes.

No treatment-related fatalities occurred. The weight-gain of the dams in the 100 mg/kg group was lower than the control animals throughout the entire pregnancy as well as during administration.

Foetal weight in the 100 mg/kg group was reduced and there was a higher incidence of runts.

There was no evidence of possible teratogenicity. 30 mg/kg produced no damage in either dams or foetuses.
Executive summary:

25 inseminated Wistar rats per group received 1,2,4-triazole on days 6 to 15 of pregnancy in daily oral doses of 0, 10, 30 or 100 mg/kg bodyweight. The dams were examined with regard to bodyweight, appearance and behaviour. The foetuses obtained by Caesarian section on day 20 of pregnancy were investigated for embryotoxic effects by weighing and by observing the external and internal morphological changes. No treatment-related fatalities occurred. The weight-gain of the dams in the 100 mg/kg group was lower than the control animals throughout the entire pregnancy as well as during administration. Foetal weight in the 100 mg/kg group was reduced and there was a higher incidence of runts. There was no evidence of possible teratogenicity. 30 mg/kg produced no damage in either dams or foetuses.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

According to the HPV dossier (US EPA 2009, 1H-1,2,4 -triazole) :

1-In an OECD Guide-line 414 "Teratogenicity" study in rats, TA was administered by oral gavage to groups of 25 successfully mated and presumably pregnant Bor: WISW (SPF Cpb) rats from day 6 to 15 of pregnancy at daily dose levels of 0, 10, 30, or 100 mg/kg bw/day (Bayer, 1989a; RCC, 2003). There were no mortalities. No treatment-related clinical signs were observed. Mean body weight gain was significantly reduced at 100 mg/kg bw. Fetal weight was reduced at 100 mg/kg bw/day. The incidence of runts was higher at 100 mg/kg bw/day. There were no treatment related malformations. The maternal NOAEL was =30 mg/kg bw/day based on retarded weight gain at 100 mg/kg bw and the developmental NOAEL was= 30 mg/kg bw/day based on an increased incidence of runts and lower fetal weights at 100 mg/kg bw/day.

2-In an OECD Guide-line 414 "Teratogenicity" study in rats, TA administered by oral gavage to groups of 25 successfully mated and presumably pregnant Bor: WISW (SPF Cpb) rats from day 6 to 15 of pregnancy at daily dose levels of 0, 100 or 200 mg/kg bw/day in the vehicle (0.5% aqueous Cremophor-EL) (Bayer, 1989b; RCC, 2003). There were no mortalities. No treatment-related clinical signs were observed. Food consumption was not affected. Mean body weight gain was slightly reduced at 100 mg/kg bw/day and markedly lower at 200 mg/kg bw/day. Fetal weight and placental weight were reduced at 100 and 200 mg/kg bw/day. The incidence of runts was higher at 100 and 200 mg/kg bw/day. The incidence of fetuses with minor skeletal deviations was higher at 100 mg/kg bw/day. The number of surviving fetuses per dam was reduced at 200 mg/kg bw/day. The incidence of fetuses with malformations (including cleft palate and hydronephrosis) was higher at 200 mg/kg bw/day. The maternal NOAEL was<100 mg/kg/day based on retarded weight gain at 100 mg/kg bw and the developmental NOAEL was <100 mg/kgbw/day based on an increased incidence of runts, lower fetal and placental weights, and a higher incidence of minor skeletal deviation at 100 mg/kg/day.

3-OECD Guide-line 414 "Teratogenicity" study in rabbits, TA was administered by gavage to groups of twenty-five time-mated New Zealand White rabbits [Hra: NZW:SPF] in 0.5% aqueous CMC at dose levels of 0, 5, 15, 30 and 45 mg/kg bw/day on days 6 though 28 (Argus, 2004). Five does in the 45 mg/kg bw/day dosage group were sacrificed due to their moribund condition. All other does survived to day 29 of gestation (GD 29). Clinical observations were noted in the 45 mg/kg bw/day group and included decreased motor activity, clear perinasal substance, ptosis, excess salivation and hyperpnoea. Most of these observations occurred in the does that were sacrificed moribund. Additional observations included scant feces, ungroomed coat, head tilt, lacrimation, flared nostrils and cold to touch. Body weight gains were reduced and gravid uterine weights were significantly reduced in the 45 mg/kg bw/day group. Fetal weights were significantly reduced in the 45 mg/kg bw/day group. There were a few alterations of the urogenital system (low set, small, absent kidneys and/or an absent ureter) which occurred in several fetuses of the maternally toxic 45 mg/kg/day dosage group. There were no other dosage-dependent and/or significant differences in the litter or fetal incidences of any gross external, soft tissues or skeletal alterations. Skeletal ossification averages per foetus per litter did not differ among the groups. The maternal NOAEL was = 30 mg/kg bw bw/day based on mortality, reduction in body weight gain, and clinical symptoms at 45 mg/kg bw/day and the developmental NOAEL was = 30 mg/kg bw/day based on lower fetal weights and a slight increase in urogenital alterations for several fetuses at 45 mg/kg bw/day. In an OECD Guide-line 414 "Teratogenicity" study, TA administered by oral gavage to groups of 25 successfully mated and presumably pregnant Bor: WISW (SPF Cpb) rats from day 6 to 15 of pregnancy at daily dose levels of 0, 100 or 200 mg/kg bw/day in the vehicle (0.5% aqueous Cremophor-EL) (Bayer, 1989b; RCC, 2003). There were no mortalities. No treatment-related clinical signs were observed. Food consumption was not affected. Mean body weight gain was slightly reduced at 100 mg/kg bw/day and markedly lower at 200 mg/kg bw/day. Fetal weight and placental weight were reduced at 100 and 200 mg/kg bw/day. The incidence of runts was higher at 100 and 200 mg/kg bw/day. The incidence of fetuses with minor skeletal deviations was higher at 100 mg/kg bw/day. The number of surviving fetuses per dam was reduced at 200 mg/kg bw/day. The incidence of fetuses with malformations was higher at 200 mg/kg bw/day. The maternal NOAEL was fetal and placental weights, and a higher incidence of minor skeletal deviations at 100 mg/kg bw.

4/In an OECD 416 “Two Generation Reproductive Toxicity” study, TA was administered continuously via the feed to Wistar Hannover rats (30/sex/dietary level) at nominal dietary concentrations of 0, 250, 500, and 3000 ppm (Bayer, 2005; RCC, 2003; TDMG, 2006). The exposure period was from 10 weeks premating P-generation through 61 days old for F2-pups. The histopathological lesions in the cerebellum were identical to that observed in the sub-chronic and neurotoxicity study in rats and are characterized by variable loss of Purkinje cells, white matter degeneration and gliosis. The white matter tract degeneration presented as nerve fiber (axonal) swelling or fragmentation often with digestion chambers containing debris and macrophage. No similar changes were noted in the 500 ppm animals. The statistical differences associated with certain parameters of F2-pups (pup weights, preputial separation, brain-and spleen weights) are considered to be attributed to an incidental difference in the time of delivery for both the 250- and 500-ppm dietary groups, relative to concurrent controls, rather than exposure to the test substance. The control pups were heavier than the treated group pups and above the range of historic controls because more control dams delivered on gestation day 23 and fewer on day 21 in comparison to the treatment groups. These results, including an extensive investigation of brain morphology, provided no evidence of developmental neurotoxicity at a dietary level of 500-ppm. The developmental NOAEL was > 500 ppm (equivalent to > 35.8 mg/kg bw/day) based on lack of treatment-related effects in F1 and F2 pups at 250 and 500 ppm.


Justification for selection of Effect on developmental toxicity: via oral route:
A NOAEL was defined in the reliable study.

Justification for classification or non-classification

1,2,4 -triazole is classified in category 2, H361 "Suspected of damaging fertility or the unborn child" according to the Regulation EC no. 1272/2008 (CLP) and classified R63 according to the EU Directive 67/548/EEC.