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Multiple mechanistic studies were conducted to elucidate the mode of action for formation of uterine adenocarcinomas in F344 rats at high doses of Afidopyropen. These studies included studies that support the purported mode of action of the test substance acting as an effective dopamine agonist that ultimately results in an increased incidence of uterine adenocarcinomas (studies with dopamine receptors, studies measuring prolactin concentrations).

Dopamine related mechanistic studies

WoE: D2 tissue binding assay 2015/1204930

- Field stimulated rabbit ear artery; D2; agonist effect; standard protocol: test substance showed D2 agonist-like effects that were not reversed by (-)Sulpiride

- Field stimulated rabbit ear artery; D2; agonist effect, modified protocol; pre-incubation with (-)Sulpiride: concentration-dependent decrease in the twitch contraction amplitude; confirmatory of D2 agonist effect

WoE: Binding assay 2015/1204929

Radioligand binding:

- dopamine receptor (D1h) agonist and antagonist effect: no activity

- dopamine transporter (h): no activity

Dopamine uptake

- Functional assay using synaptosomes from rat striatum: no activity

Tissue bioassay

- Rabbit splenic artery; D1 agonist and antagonist effect: no activity

D2 tissue bioassay

- Field stimulated rabbit ear artery; D2; agonist effect: test substance showed D2 agonist-like effects that wer enot reversed by (-)Sulpiride

- Field stimulated rabbit ear artery; D2; antagonist effect: no activity

WoE: Fuctional assay 2015/1117539

Radioligand binding

- Dopamine receptor (D2h) agonist and antagonist effect: no activity

There were also studies for alternative modes of action that did not ultimately prove to be relevant for the test substance (such as estrogen receptor agonism, enzyme induction, estrogen transcriptional activation).

WoE: Estrogen receptor transcriptional activation 2015/1245767

The test substance is not an agonist of human estrogen receptor alpha (hERα) in the HeLa-9903 model system.

WoE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol: 2015/1245768

The test substance was classified as 'equivocal', i.e. the result is ambiguous, or cannot be properly identified due to limitations of the assay.

WoE: Induction of CYP1A1 and CYP1B1 2015/1183794

This study investigated a possible mode of action for uterine tumour formation by assessing the levels of hepatic and uterine CYP1Al and CYP1B1 following 14 days test substance administration at a concentration of 3000 ppm in RM1 powder diet to 8-9 weeks old female Fischer 344 rats. Treatment of female F344 rats with the test substance via the diet at 3000 ppm resulted in small increases in liver weight and liver/bodyweight ratio. These changes were accompanied by small increases in CYP1A1 mRNA expression, EROD activity and the 2-hydroxylation of estradiol. No effects were observed on the 4-hydroxylation of estradiol or CYP1B1 mRNA expression. In the uterus, the test substance decreased uterine weight, while a moderate induction of CYP1A1 mRNA was observed. There were no increases in CYP1B1 mRNA expression.

WoE: Effect on prolactin levels in 28d rat oral study

The objective of the study was to determine treatment-related effects on prolactin concentrations after 4 weeks oral administration of the test substance via diet in comparison to the positive control Bromocriptine mesylate by gavage. The prolactin concentration in plasma on the circadian peak in the afternoon as well as after drug-induced (Metoclopramide) prolactin release from endogenous storages was determined. The analyses of prolactin concentration was performed considering the estrous cycle status of each animal.

The intent of this study and the doses used was to duplicate the dose levels used in the high dose rat cancer study (2014/1215781) and observe the effects on prolactin concentrations.

The test substance was administered orally via the diet to groups of 20 female Fischer F344 rats at concentrations of 0 (test group 0), 300 (test group 1), 1000 (test group 2) and 3000 ppm (test group 3) over a period of 4 weeks. Bromocriptine mesylate was administered orally by gavage to a group of 20 female Fischer F344 rats at a dose of 10 mg/kg bw/d (test group 4) over a period of 4 weeks. Two strategies were used to assess prolactin concentration. First, plasma samples were taken from all animals before (study day -7) and after a specific duration of exposure (in the afternoon of study day 24 and in the morning of study day 28). Second, animals only in the selected estrus cycle stage proestrous (or estrous) representing the stages with the highest plasma concentration of prolactin) were sampled and analyzed.

Clinical observations, food consumption and body weight were determined regularly during acclimatization period and at least weekly during administration period. Estrous cycle was evaluated on days -7 and -8 and from day 0 onwards. Prolactin concentration analysis was conducted on study day -7, 24 and 28 from all animals as well as from animals in the estrous cycle stages proestrus (and estrus) during parts of the administration period. After the administration period, all rats were sacrificed and assessed by gross pathology, followed by histopathological examinations of seven selected organs as well as all gross lesions.

At the 3000 ppm treatment, clinical examinations showed a decreased number of estrous cycles (0.15 in comparison to 3.7 in control) and a decreased number of animals with estrous cycle (3 out of 20). Clinical pathology showed decreased prolactin values in proestrus between study days 0 and 4 as well as between days 15 and 22. Decreased prolactin values were also observed after stimulation with metoclopramide compared to controls in rats of metestrus. Various pathology findings were present including decreased absolute and relative weight of ovaries, decreased absolute and relative uterus weight, decreased absolute and relative pituitary gland weight, decreased absolute and relative adrenal gland weight, diffuse atrophy of ovaries, diffuse atrophy of the uterus, diffuse atrophy of the cervix and diffuse atrophy of the vagina.

At the 1000 ppm treatment, clinical examinations showed no treatment-related adverse effects. Clinical Pathology showed decreased prolactin values in proestrus between study days 0 and 4 as well as between days 15 and 22. Decreased prolactin values were also observed after stimulation with metoclopramide compared to controls in rats of metestrus. There were no treatment-related, adverse effects on pathology.

At the 300 ppm Afidopyropen treatment there were no treatment-related adverse effects in any parameter.

In the treatment with Bromocriptine mesylate – positive control at 10 mg/kg bw/d, clinical examinations showed a slight body weight loss from day 0 to 7 as well as significantly increased number of estrous cycles and significantly decreased length of estrous cycles. Clinical pathology showed decreased prolactin values in proestrus between study days 0 and 3 as well as between days 16 and 22 and in estrus between study days 18 and 22. Decreased prolactin concentration was observed in estrus and metestrus in the afternoon of study day 24. Increased prolactin concentration in estrus and metestrus in the morning of study day 28 before metoclopramide stimulation. Decreased prolactin concentration values were observed after stimulation with metoclopramide compared to controls in rats of estrus and metestrus. Pathology showed an increased absolute (+49%) and relative (+47%) weights of ovaries as well as decreased absolute (-10%) and relative (-11%) weights of the pituitary gland.

Under the conditions of this study, with the administration of the test substance orally via the diet and Bromocriptine mesylate via gavage to female Fischer F344 rats for 4 weeks the NOEL (no observed effect level) was 100 ppm (test substance intake 18.2 mg/kg bw/d) based on prolactin plasma level alterations.

Both the test substance and Bromocriptine mesylate, caused a decrease of prolactin plasma levels with or without metoclopramide induced release.