Amitrole

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

October 1985 - January 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Study design is considered to follow OECD guideline Test Guideline No 414 of 1981.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

no

Principles of method if other than guideline:

In addition to the "teratology sacrifice portion" killed on gestational day 21, groups of dams were designed as the "postnatal sacrifice portion'. For these animals, pups and dams were killed on postnatal day 21.

GLP compliance:

no

Remarks:

No but according with EPA rules.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Amitrole

EC Number:

200-521-5

EC Name:

Amitrole

Cas Number:

61-82-5

Molecular formula:

C2H4N4

IUPAC Name:

amitrole

Test material form:

solid: particulate/powder

Remarks:

fine white powder

Details on test material:

- Name as cited in the report: Aminotriazole Technical

Test animals

Species:

rat

Strain:

other: CD® outbred albino rats

Remarks:

CD®-Crl: COBS® CD®(SD)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: males: 62 days old ; females: 56 days old
- Weight at study initiation: not reported
- Fasting period before study: Not reported
- Housing: Stainless steel wire-mesh cages. Females in the postnatal portion were transferred to polypropylene shoebox cages on gd 18.
- Diet (e.g. ad libitum): Certified Ground Rodent Chow(R), No. 5002, batch No. OCT 01 85 1B, Ralston Purina Co., ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 2 weeks (quarantine)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported but recorded continuously
- Humidity (%): not reported but recorded continuously
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Rats received on October 29, 1985
Sacrifice period for teratology portion: December 9 through December 12, 1985.
Sacrifice period for postnatal portion: January 7, 1986 or December 17, 1985 for dams with litters or females who failed to deliver, respectively.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized (Millipore®)

Details on exposure:

VEHICLE
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 10 mL/kg bw (administered dose volume)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of dosing formulations was performed pre- and post-dosing.
It indicated that all formulations were within 92.0 - 105.4 % of target.
The formulation were stable for at least 15 days.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: quarantine period (2 weeks)
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not reported
- Further matings after two unsuccessful attempts: not reported
- Verification of same strain and source of both sexes: not reported
- Proof of pregnancy: vaginal plug referred to as gestational day (gd) 0
- Any other deviations from standard protocol: not reported

Duration of treatment / exposure:

Day 6 through day 15 of gestation

Frequency of treatment:

Daily

Duration of test:

approximately 21 days for the teratology portion and 42 days for the postnatal portion.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

analytical concentration:
- prior to dosing period: 9.2 mg/mL (92.0% of Target)
- subsequent to dosing period: 9.4 mg/mL (94.0% of Target)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

analytical concentration:
- prior to dosing period: 52.7 mg/mL (105.4% of Target)
- subsequent to dosing period: 48.4 mg/mL (96.8% of Target)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

analytical concentration:
- prior to dosing period: 100.1 mg/mL (100.1% of Target)
- subsequent to dosing period: 92.8 mg/mL (92.8% of Target)

No. of animals per sex per dose:

38 pregnant females/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of a dose range-finding study

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily at dosing, and at 0.5 and 2 hours after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Gestational Day 0, 6, 12, 15, 18 and 21

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Gestational Day 0-3, 3-6, 6-9, 9-15, 15-18 and 18-21

WATER CONSUMPTION: Not reported

POST-MORTEM EXAMINATIONS: Yes
* Postnatal portion:
- Sacrifice on postnatal day #21 or on gestational day #24 for females which did not deliver before.
- Organs examined:
Maternal thyroids were weighed and retained in 10% buffered neutral formalin.
The pregnancy statuss of females which did not deliver by gd 24 was evaluated by examination of uterine contents or by staining apparent nongravid utero with 10% ammonium sulfide.
* Teratology portion:
- Sacrifice on gestational day #21
- Organs examined:
Gravid uterus, ovaries (including corpora lutea), cervix, vagina, peritoneal and thoracic cavities were examined grossly.
Ovarian corpora lutea of pregnancy were counted.
Maternal liver, and uterine weights were determined.
Maternal thyroids were weighed and fixed in buffered neutral 10% formalin for possible subsequent histopathologic examination.
Uteri from females that appeared nongravid were placed in a 10% ammonium sulfide solution for detection of early resorptions.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of dead and live fetuses

Fetal examinations:

- External examinations: Yes: all fetuses
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Leven's test for equal variances, ANOVA and t-test for parametric data.
Kruskal-Wallis test, Mann-Whitney U test and Fisher's Exact Test for nonparametric data.

Historical control data:

None

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable (gavage study)

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Maternal body weights exhibited no treatment-related differences across groups.
- Weight gain for gd 6-12 was slightly but statistically significantly increased at 100 and 1000 mg/kg/day, but not at 500 mg/kg/day relative to controls. These increases were apparently not treatment related.
- Weight gain was significantly reduced for gd 6-18 at 1000 mg/kg/day with a dose-related trend.
- There were no significant differences among groups for maternal body weight or weight gain during lactation period.
- No treatment-related differences in maternal body weight and corrected body weight (body weight at sacrifice minus gravid uterus weight) at the teratological sacrifice on gd 21.
Reduced weight gain may be due to reduced food consumption at 1000 mg/kg bw/day and/or effects on maternal thyroids.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Food consumption was decreased for the intervals gd 12-15, 15-18 and 15-21 at 500 and 1000 mg/kg/day.
- Food consumption during lactation was unaffected by treatment.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable (gavage study)

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

not examined

Description (incidence and severity):

Not applicable (gavage study)

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- No treatment-related differencies in gravid uterine weight or in absolute or relative liver weight.
- Absolute and relative thyroid weights were increased at 500 and 1000 mg/kg/day relative to control values in the postnatal portion and in the teratology portion to a similar degree.
Treatment-related adverse effects regarding thyroid.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Percent preimplantation and postimplantation loss were equivalent across groups.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day, one female had a totally resorbed litter at scheduled sacrifice.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no effect of treatment on viable or non-viable (early and late resorptions and dead fetuses) implantations per litter.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Pregnancy rate was high and approximately equivalent for all dose groups.

Other effects:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fetal body weight per litter were reduced at 1000 mg/kg/day relative to control values.
It may be due to effects on fetal thyroids and/or reduced food consumption of dams and/or effects on maternal thyroids.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

All litters (except for one at 500 mg/kg bw/day in the teratology portion) had one or more live fetuses at scheduled teratology sacrifice or at delivery.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

There was no significant differences among all groups in the incidence of individual external malformations.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

A total of 135 different types of fetal skeletal variations were observed.
Of these, six exhibited a significantly different incidence at 1000 mg/kg bw/day relative to that of controls. These findings included increased incidences of unossified cervical centrum 6, of poorly ossified maxillary bones, and of enlarged biparietal suture in the skull.
There were also decreased incidences (relative to controls) of bilobed cervical centra 1-3 and/or 4, and of some (1-4) proximal phalanges of the hindlimb poorly ossified and unossified.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

There was no significant differences among all groups in the incidence of individual visceral malformations (including cranofacial).
However, the incidence of one visceral finding, out of 54 types of visceral variations observed, that of dark thyroids, was significantly increased at 1000 mg/kg bw/day relative to that of controls. In addition, the incidence of enlarged fetal thyroids was increased at 500 and 1000 mg/kg bw/day but these values were not statistically significantly different from the incidence in control fetuses.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

visceral malformations

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

See 'Attached background material' for tables of results.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, NOAEL (maternal toxicity) = 100 mg/kg bw/day, based on reduced bodyweight gain, reduced food consumption and enlarged maternal thyroids at 500 and 1000 mg/kg bw/day.
NOAEL (developmental toxicity) = 100 mg/ kg bw/day, based on enlarged and dark fetal thyroids observed at 500 and 1000 mg/kg bw/day (not statistically significant at 500 mg/kg bw/day).
The classification of the substance as STOT-RE 2 (H373: May cause damage to organs through prolonged or repeated exposure) for thyroid according to the Regulation (EC) No. 1272/2008 (CLP) is confirmed.

Executive summary:

In a teratogenicity study, test substance was daily administered by gavage at dose levels of 0, 100, 500, or 1000 mg /kg bw/day in deionized water to female CD® rats on gestational days (gd) 6 to 15.

24 out of 38 plug-positive females per group were employed for the teratology sacrifice portion. The remaining 14 plug-positive females per group were employed for the postnatal sacrifice portion.

Clinical observations were taken daily and maternal body weights were taken on all study females on GD 0, 6, 12, 15 and 18. Food consumption was also mesured on all females for the intervals GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21. At scheduled teratology necropsy on gd 21, the approriate dams were evaluated for body weight, liver and thyroid weight, gravid uterine weight and status of implantation sites. Live fetuses were dissected from the uterus, counted, weighed, sexed and examinated for external abnormalities. Approximately one-half of the live fetuses in each litter were examined for visceral malformations. The fetuses were decapitated and the heads were examined for soft tissue craniofacial malformations. The remaining fetuses in eac litter were eviscerated and examined for skeletal defects and deficits.

The females from the postnatal portion were allowed to litter. These dams and their litters were weighed and examined on postnatal day 0, 4, 7, 14 and 21; food consumption was mesured for the intervals postnatal days 0-4, 4-7, 7-10 and 10-14. Each litter was culled to eight pups on postnatal day 4 and sacrificed after all observations on postnatal day 21. At postnatal sacrifice maternal thyroids were weighed.

There were no treatment-related maternal deaths. Slight maternal toxicity was indicated by reduced weight gain for GD 6 -18 at 1000 mg/kg bw/day but not prior to or subsequent to this period, and reduced food consumption for gd 12 -15, 15 -18 and gd 15 -21 at 500 and 1000 mg/kg bw/day. There were no differences among groups for gestation or lactation maternal body weights or for lactational weight gain or food consumption. No treatment-related clinical signs were observed. At the gd 21 sacrifice there were no effects of treatment on maternal body weight, gravid uterine weight, or on absolute or relative liver weight. Maternal thyroid weight was increased at 500 and 1000 mg/kg bw/day. Reproductive parameters, including ovarian corpora lutea of pregnancy, total, viable and nonviable implantations per litter and sex ratio were unaffected by treatment. Fetal body weight per litter was reduced at 1000 mg/kg bw/day.

There was no significant increase in the incidence of malformations in any treated group relative to controls and no differences among groups for pooled external, visceral, skeletal or total variations. At 1000 mg/kg bw/day there were seven variations with incidences different from that of controls, including dark fetal thyroids and indications of reduced ossification. Enlarged and/or dark fetal thyroids also exhibited an increased incidence at 500 and 1000 mg/kg bw/day relative to that in controls.

Postnatal observations indicated no differences among groups for any pup parameters at any time points evaluated, including litter sizes, sex ratio, pup weights per litter or survival indices. Maternal thyroid weights were still increased at 500 and 1000 mg/kg bw/day at postnatal sacrifice.

Under the test condition, administration of test material by gavage to CD® rats during organogenesis resulted in evidence of maternal and fetotoxicity toxicity at 500 and 1000 mg/kg bw/day. Postnatal evaluations indicated all observed maternal and perinatal effects were transient except for enlarged maternal thyroids. No teratogenicity was observed at any dose level.

Therefore, the NOAEL for maternal and developmental toxicity is considered to be 100 mg/kg bw/day and the classification of the substance as STOT-RE 2 (H373: May cause damage to organs through prolonged or repeated exposure) for thyroid according to the Regulation (EC) No. 1272/2008 (CLP) is confirmed.