1H-1,2,4-triazole, sodium salt

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

JULY 2004-FEB 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The GLP-study was performed according to internationally accepted guidelines (OPPTS Guideline No. 870.3700 (1998): Prenatal Developmental Toxicity Study; OECD Guideline No. 414 (2001): PrenatalDevelopmental Toxicity Study; JMAFF Guideline: 59 Nohsan No. 4200: Guidance on Toxicology Study Data for Application of Agricultural Chemical Registration). All study parameters are well documented and are based on the specific guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

The GLP-study was performed according to internationally accepted guidelines
(OPPTS Guideline No. 870.3700 (1998): Prenatal Developmental Toxicity Study;
OECD Guideline No. 414 (2001): PrenatalDevelopmental Toxicity Study; JMAFF
Guideline: 59 Nohsan No. 4200: Guidance on Toxicology Study Data for
Application of Agricultural Chemical Registration). All study parameters are well
documented and are based on the specific guidelines.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2,4-triazole

EC Number:

206-022-9

EC Name:

1,2,4-triazole

Cas Number:

288-88-0

Molecular formula:

C2H3N3

IUPAC Name:

1H-1,2,4-triazole

Test material form:

other: white solid flakes

Specific details on test material used for the study:

Name of test material (as cited in study report): 1,2,4-Triazole- Substance type: organic- Physical
state: white flakes- A Storage condition of test material: room temperature

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Covance Research Products, Inc., Denver, Pennsylvania- Age at study
initiation: 5.5 month- Weight at study initiation: 2.7 - 4.4 kg- Fasting period before study: no- Housing:
individually housed in units of six to eight stainless steel cages- Diet (e.g. ad libitum): Certified Rabbit
Chow #5322 (PMI Nutrition International, St. Louis, Missouri) 150 g/rabbit up to dosing, thereafter 180
- 185 g/rabbit- Water (e.g. ad libitum): Local water that had been processed by passage through a
reverse osmosis membrane ad libitum. Chlorine was added to the processed water as a bacteriostat.-
Acclimation period: The female rabbits were naturally bred by breeder male rabbits of the same source
and strain before shipment to the Testing Facility. The rabbits were mated on five consecutive days
and shipped to the Testing Facility to arrive on DGs 1, 2, 3, 4 or 5. The day of mating was considered
to be DG 0.DG= day of gestationENVIRONMENTAL CONDITIONS- Temperature (°C): 16°C to 22°CHumidity
(%): targeted at 30 % to 70 %- Air changes (per hr): 10/hr- Photoperiod (hrs dark / hrs light):
12-hours light:12-hours dark fluorescent light cycleIN-LIFE DATES: From: 2004-08-06 To: 2004-09-03

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analyses from the first and last weeks of dosing indicated that the dosing solutions were
within ±10 % of the target concentration. Homogeneity and stability of the test substance in the vehicle
were established prior to the start of dosing.

Details on mating procedure:

The female rabbits were naturally bred by breeder male rabbits of the same source and strain before
shipment to the Testing Facility. The rabbits were mated on five consecutive days and shipped to the
Testing Facility to arrive on days of gestaition (DG) 1, 2, 3, 4 or 5. The day of mating was considered
to be DG 0.DG= day of gestation

Duration of treatment / exposure:

days 6 through 28 of gestation

Frequency of treatment:

Once daily

Duration of test:

Rabbits were sacrificed on gastation day 29.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

45 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 rabbits/group

Control animals:

yes

Details on study design:

Dose selection rationale: Dosages were selected on the basis of a dosage-range developmental
toxicity study.- Rationale for animal assignment (if not random): computer-generated (weight-ordered)
randomization procedure

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: Rabbits were observed for viability at least twice
each day of the study and for general appearance at least once during the predosage period. The
rabbits were also examined for clinical observations, abortions, premature deliveries and deaths
before dosage,approximately 120 minutes after administration, at the end of the normal working day
and on the day of sacrifice.DETAILED CLINICAL OBSERVATIONS: Yes / No / No data- Time
schedule:BODY WEIGHT: Yes - Time schedule for examinations: Body weights were recorded on DG
0 (day of gestation), the day of arrival at the Testing Facility, dailyduring the dosage period and on the
day of sacrifice.FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Feed
consumption values were recorded daily after arrival at the Testing Facility.WATER CONSUMPTION
AND COMPOUND INTAKE (if drinking water study): No POST-MORTEM EXAMINATIONS: Yes-
Sacrifice on gestation day 29- Organs examined: Caesarean-sectioned, and a gross necropsy of the
thoracic, abdominal and pelvic viscera was performed.OTHER:Rabbits that were sacrificed because of
moribund condition were examined for the cause of the moribund condition and for gross lesions on
the day the observation was made.Pregnancy status and uterine contents were recorded. Concepti in
utero were examined to the extent possible, using the same methods described for term fetuses.
Ovaries and uterine content
Uteri of apparently nonpregnant rabbits were examined after staining with 10 % ammonium sulfide to
confirm the absence of implantation sites.The number and distribution of corpora lutea were recorded.
The uterus of each rabbit was excised, weighed and examined for pregnancy, number and distribution
of implantation sites, early and late resorptions and live and dead fetuses. An early resorption was
defined as one in which organogenesis was not grossly evident. A late resorption was defined as one
in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term
fetus that responded to stimuli. Nonresponding term fetuses were considered to be dead. Dead
fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme
autolysis indicated that the fetus was a late resorption. Placentae were examined for size, shape and
color.

Ovaries and uterine content:

Ovaries and uterine content
Uteri of apparently nonpregnant rabbits were examined after staining with 10 % ammonium sulfide to
confirm the absence of implantation sites.The number and distribution of corpora lutea were recorded.
The uterus of each rabbit was excised, weighed and examined for pregnancy, number and distribution
of implantation sites, early and late resorptions and live and dead fetuses. An early resorption was
defined as one in which organogenesis was not grossly evident. A late resorption was defined as one
in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term
fetus that responded to stimuli. Nonresponding term fetuses were considered to be dead. Dead
fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme
autolysis indicated that the fetus was a late resorption. Placentae were examined for size, shape and
color.

Fetal examinations:

Each fetus was removed from the uterus, placed in an individual container and individually identified
with a tag noting the study number, litter number and uterine distribution. Each fetus was subsequently
weighed and examined for gross lesions. Live fetuses were sacrificed by an intraperitoneal injection of
EUTHASOL®, Diamond Animal Health, Inc., Des Moines, Iowa. All fetuses were examined internally
to identify sex.Cavitated organs were evaluated in all fetuses by dissection. A single cross-section was
made between the parietal and the frontal bones of approximately one-half of the fetuses per litter,
and the brain was examined in situ. The remaining fetuses were decapitated, and the heads were
fixed in Bouin’s solution and examined for soft tissue alterations (including eyes, brain, nasal passages
and tongue) using Wilson’s sectioning technique. All fetuses were eviscerated, cleared, stained with
alizarin red S and examined for skeletal alterations. The fetuses were initially fixed in alcohol. Skeletal
preparations were retained in glycerin, with thymol added as a preservative.

Statistics:

Clinical observations and other proportion data were analyzed using the Variance Test for
Homogeneity of the Binomial Distribution.Continuous data (e.g., maternal body weights, body weight
changes, feed consumption values and litter averages for percent male fetuses, percent resorbed
conceptuses, fetal body weights, fetal anomaly data and fetal ossification site data) were analyzed
using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e.,
Bartlett’s Test was not significant (p>0.001)]. If the Analysis of Variance was significant (p=0.05),
Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis
of Variance was not appropriate [i.e., Bartlett’s Test was significant (p=0.001)], the Kruskal-Wallis Test
was used,when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test
was statistically significant (p=0.05), Dunn’s Method of Multiple Comparisons was used to identify the
statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test
was used to analyze the data.Count data obtained at Caesarean-sectioning of the dams were
evaluated using the procedures described above for the Kruskal-Wallis Test.

Historical control data:

Data of 74 studies with 967 rabbits provided within the study report ( Jan. 2002 - Jan. 2004)
Results and

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Five does in the 45 mg/kg/day dosage group were sacrificed due to their moribund condition.Adverse
clinical observations related to the test substance occurred only in the 45 mg/kg/day dosage group.
The number of does with decreased motor activity, clear perinasal substance, ptosis, excess salivation
and hypernea was significantly increased in this dosage group.Most of these observations occurred in
the does that were sacrificed moribund.Additional observations considered related to the highest dose
of the test substance or interrelated with the moribund condition of the doe included: scant feces,
ungroomed coat, head tilt, lacrimation, flared nostrils and cold to touch.Other clinical observations
were not considered related to the test substance because:1) the number of does affected was not
dosage-dependent; 2) no more than three does in any group had the observation; and/or 3) the
observations are common in this strain of rabbit at this Test Facility. These observations included
ungroomed coat (except in the 45 mg/kg/day dosage group), scant feces, (except in the 45 mg/kg/day
dosage group), soft or liquid feces, localized alopecia on the limbs, scab on the back, sparse coat, red
perioral substance, red perivaginal substance and a red substance in the cage pan.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gains
were significantly reduced for DGs 9 to 12, 21 to 24 and for the entire dosage period (calculated as
DGs 6 to 29) and the entire study period (DGs 0 to 29) in the 45 mg/kg/day dosage group. Body
weights did not significantly differ among the other groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fetal weights (male, female and total) were significantly reduced in the 45 mg/kg/day dosage group.
No other Caesarean-sectioning or litter parameters were affected by dosages of the test substance as
high as 45 mg/kg/day. T
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Fetal weights (male, female and total) were significantly reduced (p=0.05 to p=0.01) in the 45
mg/kg/day dosage group. No other Caesarean-sectioning or litter parameters were affected by
dosages of the test substance as high as 45 mg/kg/day. T

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Pregnancy occurred in 24 to 25 does in each dosage group. In addition to the does that were
sacrificed due to their moribund condition, one doe (8123) in the 45 mg/kg/day dosage group delivered
on DG 29. This doe had 15 implantation sites with 11 fetuses and one late resorption in utero, one
aborted fetus and two late resorptions. Caesareansectioning observations were based on 25, 24, 24,
25 and 19 pregnant does with one or more live fetuses in the 0 (Vehicle), 5, 15, 30 and 45 mg/kg/day
dosage groups,respectively.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

he litter averages for corpora lutea,
implantations, litter sizes, live fetuses, early and late resorptions, percent resorbed conceptuses, and
percent live male fetuses were comparable among the five dosage groups and did not significantly
differ. No doe had a litter consisting of only resorbed conceptuses. There was one dead fetus in the 30
mg/kg/day dosage group. All placentae appeared normal.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

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 fetus per litter did not differ among the groups.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

visceral malformations

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

visceral/soft tissue: urinary

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

45 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

Applicant's summary and conclusion

Conclusions:

On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) of 1,2,4-Triazole
is 30 mg/kg/day (the 45 mg/kg/day dosages caused mortality, adverse clinical observations and
reductions in body weight gain). The developmental NOAEL is also 30 mg/kg/day. The 45 mg/kg/day
dosage caused a reduction in fetal weights and a slight increase in urogenital alterations for several
fetuses.

Executive summary:

One hundred twenty-five time-mated [Hra: (NZW)SPF] rabbits were randomly assigned to five dosage

groups, 25 rabbits per group. The test substance, 1,2,4-¿riazole, CAS Number 288-88-0 and/or the

vehicle, Aqueous 0.5% (w/w) carboxymethylcellulose (CMC) was administered to the rabbits orally

(stomach tube) once daily on days 6 through 28 (DGs 6 through 28) at dosages of 0, 5, 15, 30 and 45

mg/kg/day. The dosage volume was 10 mL/kg, based on individual body weights recorded daily before

administration. Viabilities, clinical observations, body weights and feed consumption values were

recorded. Rabbits were sacrificed on DG 29, Caesarean-sectioned and examined for the number and

distribution of corpora lutea, implantation sites and uterine contents. A gross necropsy of the thoracic,

abdominal and pelvic viscera was performed. Fetuses were weighed and examined for gross external,

visceral and skeletal alterations and sex.

Five does in the 45 mg/kg/day dosage group were sacrificed due to their moribund condition. All other

does survived to day 29 of gestation (DG 29). Adverse clinical observations related to the test

substance occurred only in the 45 mg/kg/day dosage group. The number of does with decreased

motor activity, clear perinasal substance, ptosis, excess salivation and hypernea was significantly

increased in this dosage group. Most of these observations occurred in the does that were sacrificed

moribund. Additional observations considered related to the highest dose of the test substance or

interrelated with the moribund condition of the doe included: scant feces, ungroomed coat, head tilt,

lacrimation, flared nostrils and cold to touch. Body weight gains were significantly reduced for DGs 9 to

12, 21 to 24 and for the entire dosage period (calculated as DGs 6 to 29) and the entire study period

(DGs 0 to 29) in the 45 mg/kg/day dosage group. Body weights did not significantly differ among the

other groups. Gravid uterine weights were significantly reduced in the 45 mg/kg/day dosage group. No

other statistically significant differences occurred among the groups for either body weight gains,

corrected body weights (body weight on DG 29 minus the gravid uterine weight) or corrected body

weight gains. Absolute and relative feed consumption values were unaffected by dosages of the test

substance as high as 45 mg/kg/day.

Fetal weights (male, female and total) were significantly reduced in the 45 mg/kg/day dosage group.

No other Caesarean-sectioning or litter parameters were affected by dosages of the test substance as

high as 45 mg/kg/day. 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 fetus per litter did not differ among the groups.