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Key value for chemical safety assessment

Effects on fertility

Description of key information

Two-generation reproductive toxicity in rats (OECD416, GLP, Rel.2):

NOAEL(parental toxicity) = 2 ppm (eq. to 0.12 mg/kg bw/day) ;

NOAEL(reproductive toxicity) = 15 ppm (eq. to 0.9 mg/kg bw/day)

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
0.12 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is GLP-compliant and of high quality (Klimisch score = 2).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a two-generation reproduction toxicity study was performed in rats according to the OECD guideline 416 and in compliance with GLP, the test substance was given continuously to 5 groups of F0 Sprague-Dawley rats (30/sex/dose) by admixture to the diet at the dose levels of 0.5, 2, 15 and 112.5 ppm. The F0 control animals (30 males and 30 females) received the untreated diet only. After 72 days of treatment in males and females, the F0 male and female rats were paired. Treatment was continued in males until weaning of the F1 offspring and in females during the same period. F1 pups were paired when they were 12 to 14 weeks old. The F1 females were allowed to deliver normally. Macroscopic examination of F0 males and females, F1 males and females, F1 and F2 pups was performed. Organ weights and microscopic examination were performed on a range of tissues.

Under the experimental conditions, the test substance was not toxic for F0 and F1 parent animals up to 2 ppm and did not induce adverse effects on the reproductive performance of F0 and F1 animals, nor impaired the development of F1 and F2 off-springs up to 15 ppm. The dose of 15 ppm was toxic for the F0 (males only) and F1 parental animals, as shown by minimal increased thyroid activity. The 112.5 ppm dose level was severely toxic for F0 and F1 parents, impaired their fertility and disturbed the development of F1 and F2 litters. Nevertheless, all findings on reproduction correlated with severe systemic parental toxicity, so that a primary effect on reproduction is excluded.

NOAEL(parental toxicity) = 0.12 mg/kg bw/day (2 ppm)

NOAEL(reproductive toxicity) = 0.9 mg/kg bw/day (15 ppm)

Effects on developmental toxicity

Description of key information

- developmental toxicity in rats (OECD414, EPA rules, Rel.2): NOAEL(maternal and developmental toxicity) = 100 mg/kg bw/day based on effects on thyroid which are already covered by the harmonised classification STOT-RE 2 (H373) determined from the repeated dose toxicity studies.

- developmental toxicity in rabbits (OECD414, EPA rules, Rel.2): NOAEL(maternal and developmental toxicity) = 4 mg/kg bw/day and classification Cat.2 (H361d) for reproductive toxicity

- developmental toxicity in rabbits (OECD414, GLP, Rel.2): NOAEL(maternal and developmental toxicity) = 20 mg/kg bw/day

- maternotoxicity in rabbits (No guideline, GLP, Rel.1): NOAEL(maternal toxicity) = 3 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
3 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The available information as a whole meets the tonnage driven data requirement of REACH.
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

Test N° Test guideline and reliability Species (strain) Test concentration Type of exposure Conclusion
1 (Tyl, 1986a) OECD414 (version of 1981) with additional animals sacrificed on postnatal day 21 Rats (CD®-Crl: COBS® CD®(SD)BR) 100, 500, 1000 mg/kg bw/day gavage NOAEL(maternal) = 100 mg/kg bw/day
NOAEL(dev) = 100 mg/kg bw/day
STOT-RE 2 H373 (thyroid)
2 (Tyl, 1986b) OECD414 (version of 1981) Rabbits (NZW) 4, 40, 400 mg/kg bw/day gavage NOAEL(maternal) = 4 mg/kg bw/day
NOAEL(dev) = 4 mg/kg bw/day
Cat.2 reprotox H361d
3 (Kolb, 1994) OECD414 (version of 1981) Rabbits (Himalayan) 5, 20 and 80 mg/kg bw/day gavage NOAEL(maternal) = 20 mg/kg bw/day
NOAEL(dev) = 20 mg/kg bw/day
4 (Richard, 2000) Maternotoxicity study Rabbits (NZW) 3, 15, 75 mg/kg bw/day gavage NOAEL(maternal) = 3 mg/kg bw/day

Test N°1:

In an teratogenicity study performed in 1986, test substance was daily administered by gavage at dose levels of 0, 100, 500, or 1000 mg /kg body weight/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 performed at gd 21 and the remaining 14 plug-positive females per group were allowed to litter and employed for the postnatal sacrifice portion performed at postnatal day 21.

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.

NOAEL (maternal and developmental toxicity) = 100 mg/kg bw/day

STOT-RE 2 (H373) based on effects on thyroid (see section 7.5 Repeated dose toxicity)

Test N°2:

Test substance was also tested in rabbits in the same period the same lab as for Study N°1. Test substance was daily administered by gavage at dose levels of 0, 4, 40, or 400 mg /kg bw/day in deionized water to female New Zealand White rabbits on gestational days (GD) 6 to 18.

Twenty two mated females per group were employed.

There were no treatment-related maternal deaths. Maternal toxicity at 40 and 400 mg/kg bw/day was indicated by an increase in the incidence of spontaneous abortions at both dosage levels, and by a reduction in body weight and weight gain during and after the dosing period. Gravid uterine weight and absolute liver weight were unaffected by treatment, but liver weight relative to body weight was increased at 400 mg/kg bw/day. The number of ovarian corpora lutea per doe, of total implantations per litter was increased and the percent live fetuses per litter was significantly decreased at 400 mg/kg bw/day. Fetal body weight per litter was significantly reduced at 400 mg/kg bw/day.

At 40 and 400 mg/kg bw/day there were significant increases in the incidence of individual malformations, of malformations by category (external, visceral includng craniofacial, and skeletal) and of total malformations. The malformations involved the head, limbs, vertebral column and tail. There were no treatment-related effects on the incidence of individual fetal external variations. The incidence of a number of individual visceral (including craniofacial) and skeletal variations was increased at 40 and 400 mg/kg bw/day. The incidence of variations by category and of total variations was increased at 40 and 400 mg/kg bw/day.

Under the test condition, administration of test material by gavage to New Zealand White rabbits during organogenesis resulted in evidence of maternal and embryo/fetotoxicity including teratogenicity at 40 and 400 mg/kg bw/day.

NOAEL (maternal and developmental toxicity) = 4 mg/kg bw/day

Cat. 2 (H361d) for Reproductive toxicity

Test N°3:

An oral teratogenic study was conducted according to OECD Guideline No. 414 (Teratogenicity), EPA OPP 83.3 (Prenatal developmental) and EC guideline (Comission Directive 88/302/EEC) in compliance with GLP to evaluate the potential toxic effects of the test substance on the pregnant female and fetuses rabbits.

Sixteen female Russian rabbits were daily treated orally by gavage with Amitrole from day 6 to18 p.c. in doses of 0, 5, 20, and 80 mg/kg bw, respectively. On the 29th day of gestation the fetuses were delivered by cesarian section. Investigations were performed on general tolerance of the test compound by the dams as well as on its effect on intrauterine development.

Appearance, behavior, and mortality were not affected by the treatment up to and including the dose of 80 mg/kg bw/day. Macroscopic signs of compound related organ damage at necropsy were not evident up to and including 80mg/kg bw/day. Body weight gain and feed consumption in the 80 mg/kg group were reduced mainly during the first week of treatment. The water in take was decreased and excretory products (smallscybala) were affected at 80 mg/kg bw/day.

With respect to intrauterine development, the gestation rate, the resorption rate, the number of fetuses and fetal sex as well as the weight and appearance of the placentas were unaffected by the treatment up to and including 80 mg/kg bw/day.

The male fetal weight was marginally reduced at the dose of 80 mg/kg bw/day.

External, visceral and skeletal examination of the fetuses revealed no effects of Amitrole on intrauterine development up to and including the dose of 20 mg/kg bw/day. At the dose of 80 mg/kg a slight increase in skeletal variations was observed (fusions of sternebrae). A teratogenic potential of Amitrole was not evident.

Under the test conditions:

- the maternal NOAEL is 20 mg/kg bw/day, based on reduced body weight gain and food/water consumption observed at the high dose (80 mg/kg bw/day).

- the fetal NOAEL is 20 mg/kg bw/day, based on the skeletal variations (fusions of sternebrae) observed at the high dose (80 mg/kg bw/day).

Test N°4:

An oral prenatal developmental toxicity study was conducted in compliance with GLP to evaluate the potential toxic effects (mainly on thyroid function) of the test substance on the pregnant female rabbit.

Three groups of mated female rabbits (10/dose) of the KBL New Zealand White strain, received the test substance in aqueous solution, by oral gavage, at 0 (purified water), 3, 15 or 75 mg/kg bw/day, daily from Day 6 to Day 18 post-coitum, inclusive and were sacrified on day 29 post-coitum. Foetuses were only submitted to an external examination.

No treatment-related clinical signs were observed at any dose-level. There were no deaths ascribed to treatment with the test substance. There was no abortion in any group. The body weight gain, the food and water consumption were similar in the control and the 3 and 15 mg/kg bw/day groups. In the 75 mg/kg bw/day group, the body weight gain, food consumption and water consumption were lower than that of the control group (body weight gain: -66%, p < 0.01; food consumption: -37%, p < 0.001; water consumption: -24%). In blood chemistry, there were no notable changes at 3 and 15 mg/kg bw/ day. At 75 mg/kg bw/day, there was a higher cholesterol level and a lower level of the total thyroid hormones T3 and T4 (cholesterol: +225%, p < 0.01; T3: -51%, p < 0.01; T4: -29%, p < 0.01). All the hysterectomy parameters were similar in the control and the treated groups: pre- and post-implantation loss, number of foetuses and foetal weight. There were no treatment-related external malformations or variations in the foetuses of the 3 and 15 mg/kg bw/day groups. In the 75 mg/kg bw/day group, 3/66 foetuses displayed a domed-head, probably related to dilatation of cerebral ventricles/hydrocephaly. A relationship to treatment with the test substance cannot be ruled out. In dams, there were no notable differences in thyroid weights between the control and treated females, and there were no macroscopic changes (including in the thyroids) that were attributed to treatment with the test substance. The severity of the morphological changes indicative of thyroidal hyperactivity was similar between the dams given 3 mg/kg bw/day and the control dams. However, slightly higher severity was seen in the treated dams given 15 or 75 mg/kg bw/day. There were no signs of hyperactivity in the thyroids of the foetuses of any control or treated groups.

Under the test conditions, the test substance when administered to pregnant female rabbits, daily from Day 6 to Day 18 post-coitum, at 3, 15 or 75 mg/kg bw/day was clearly maternotoxic at 75 mg/kg bw/day. There were signs of slight thyroidal hyperactivity at 15 and 75 mg/kg bw/day, among the dams.

NOAEL (maternal toxicity) = 3 mg/kg bw/day

CONCLUSION:

In the developmental toxicity study in rabbits (test N°2), malformations of the head and limbs were observed. In a recent study focussing on maternotoxicity in rabbits (test N°4), in which only external examination of the foetuses was performed, malformations of the head were observed.

According to a weight of evidence approach for developmental toxicity and the conclusion on the peer review of the pesticide risk assessment of the active substance amitrole (EFSA, Journal 2014; 12(7):3742), the NOAEL of 3 mg/kg bw/day for maternal and developmental toxicity in rabbits and the harmonised classification as Cat.2 (H361d) is confirmed for AMITROLE.

Justification for classification or non-classification

Harmonized classification:

The substance has an harmonized classification in Cat.2 (H361d: Suspected of damaging the unborn child) for reproductive toxicity according to the Regulation (EC) No 1272/2008.

Self-classification:

Developmental toxicity was reported in reliable studies. As the substance already has an harmonized classification, no addition self-classification is proposed.