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EC number: 604-636-5 | CAS number: 148477-71-8
Toxicological information
Specific investigations: other studies
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Administrative data
Link to relevant study record(s)
- Endpoint:
- mechanistic studies
- Remarks:
- Study observing inhibition of enzymes involved in steroidogenesis.
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- In order to investigate which step of the biosteroid synthesis was affected, in this investigation potential inhibition of microsomal cytochrome P-450-dependent monooxygenases involved in steroid hormone synthesis, i.e. steroid 17a-monooxygenase and C-17, 20-lyase, was studied in vitro. Investigations focused mainly on parent compound and its prominent plasma metabolite Spirodiclofen-enol. Rat testicular microsomes served as an enzyme source. Microsomes were incubated with precursor steroid in the presence of test compounds and resultant products were quantified by UV spectroscopy following HPLC separation from educts. The reference inhibitor ketoconazole was included as a positive control in this study.
- GLP compliance:
- no
- Remarks:
- non-standard study type
- Type of method:
- in vitro
- Endpoint addressed:
- other: steroid synthesis
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Hsd Cpb
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- Rat testes were used in this in-vitro study as an enzyme source; Pooled testes from 10-week-old to 13-week-old untreated rats (Wistar Hsd Cpb, Harlan-Winkelmann, Borken, FRG) were used as a starting material. After removal of the capsule, testicular tissue was kept deep-frozen until used for the preparation of microsomes.
- Route of administration:
- other: In-vitro study (not applicable)
- Details on results:
- Results: No inhibition of steroid 17a-monooxygenase occurred in the presence of 50 µM Spirodiclofen (maximally employable concentration as indicated by some precipitation of test compound) or 300 µM Spirodiclofen-enol. In contrast, the reference inhibitor ketoconazole inhibited the enzyme already at 3 µM. Marginal, irrelevant effects on C-17,20-lyase were observed in the presence of 50 µM Spirodiclofen and 300 µM Spirodiclofen-enol, respectively, whereas the reference inhibitor ketoconazole strongly inhibited the enzyme at 10 µM. Inhibitory effects on these cytochrome-P-450-dependent microsomal monooxy-genases in Spirodiclofen-treated animals are unlikely to occur: Spirodiclofen could not be detected in the plasma of Spirodiclofen-treated animals, whereas Spirodiclofen-enol, which was present in micromolar concentrations, did not cause a relevant inhibition of these enzymes in vitro.
- Conclusions:
- The microsomal monooxygenases were not affected by Spirodiclofen and its metabolites so
that the inhibition of steroid synthesis was probably not mediated by these enzymes. - Endpoint:
- mechanistic studies
- Remarks:
- A study observing inhibition of biosteroid synthesis in rat enzymes.
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- In order to investigate whether treatment with Spirodiclofen interfered with microsomal dehydrogenases involved in steroid hormone biosynthesis, the effects of Spirodiclofen and its metabolites Spirodiclofen-enol, 3-OH-Spirodiclofen-enol and 4-OH-Spirodiclofen-enol on 3ß-hydroxysteroid dehydrogenase-D4, 5-isomerase and 17ß-hydroxysteroid dehydrogenase were studied in vitro. High-performance liquid chromatography was used to separate steroid precursors and products of the assays, rat testicular microsomes served as an enzyme source.
- GLP compliance:
- no
- Remarks:
- non-standard study type
- Type of method:
- in vitro
- Endpoint addressed:
- other: biosteroid synthesis
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Hsd Cpb
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- Rat testes were used in this in-vitro study as an enzyme source; Pooled testes from 10-week-old to 13-week-old untreated rats (Wistar Hsd Cpb, Harlan-Winkelmann, Borken, FRG) were used as a starting material. After removal of the capsule, testicular tissue was kept deep-frozen until used for the preparation of microsomes.
- Route of administration:
- other: In-vitro study (not applicable)
- Details on results:
- In the presence of 25 µM Spirodiclofen and 50 µM Spirodiclofen(maximally employable concentration as indicated by some precipitation of test compound), 3ß-hydroxy-steroid dehydrogenase-D4, 5 isomerase was inhibited by 20 % and 22 %, respectively. Spirodiclofen-enol up to 300 µM, 100 µM 3-OH-Spirodiclofen-enol and 100 µM 4-OH-Spirodiclofen-enol did not inhibit the enzyme. For the reference inhibitor 17a-OH-pregnenolone, a competing substrate, an IC50 value of 50 µM was established. No inhibition of 17ß-hydroxysteroid dehydrogenase occurred in the presence of 50 µM Spirodiclofen, 100 µM Spirodiclofen-enol, 1000 µM 3-OH-Spirodiclofen-enol or 1000 µM 4-OH-Spirodiclofen-enol, whereas the IC50 value for the reference inhibitor coumestrol was 4 µM. Inhibition of 3ß-hydroxy-steroid dehydrogenase-D4,5-isomerase by Spirodiclofen may contribute to the observed reduction of testosterone synthesis in cultured, Spirodiclofen-treated testicular tissue. Inhibitory effects on these dehydrogenases in Spirodiclofen-treated animals are unlikely to occur: Spirodiclofen, 3-OH-Spirodiclofen-enol and 4-OH-Spirodiclofen-enol could not be detected in the plasma of Spirodiclofen-treated animals, whereas Spirodiclofen-enol that was present in micromolar concentrations, did not inhibit these enzymes in vitro.
- Conclusions:
- A relevant effect on the microsomal dehydrogenases and, thus, a role in the inhibition of the biosteroid synthesis was not evident from this study.
- Endpoint:
- mechanistic studies
- Remarks:
- Effects of test material and its metabolites on the human estrogen and androgen receptor in vitro
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Cells were seeded in a density of 1E+5 cells per well in a opaque or transparent 96 well plate. The opaque plates were used for the reporter gen assay, the transparent one for the neutral red assay. The medium has to be completely phenyl red free for both cell cultures. Additionally, the MVLN cells were also adapted to DCC - FCS to increase their hormonal sensitivity. The cells were cultivated over 3 days under these conditions before the compounds were tested. As a positive control for the estrogen system, 17ß-estradiol was used for the estrogenic activity and hydroxytamoxifen for the antiestrogenic activity. For the androgen system, methyltestosteron and flutamide was used.
- GLP compliance:
- no
- Remarks:
- non-standard study type
- Type of method:
- in vitro
- Endpoint addressed:
- other: androgenic or anti-androgenic potential
- Species:
- other: human
- Strain:
- other: PALM cells
- Sex:
- not specified
- Details on test animals or test system and environmental conditions:
- For this assay PALM cells were used; they were obtained from Prof. Nicolas, INSERM U439 (Montpellier, France). The PALM cells were cultivated in Nutrient Mixture F12 (HAM) supplemented with 7 % DCC - FCS (dextran coated charcoal) and 1 % Penicillin/ Streptomycin solution (all Gibco, Eggenstein). They were maintained in the cell incubator with humidified air and a CO2 concentration of 7.5 %. The cells were seeded in a density of 1E+5 cells per well in a opaque or transparent 96 well plate. The opaque plates were used for the reporter gen assay, the transparent ones for the neutral red assay. The medium had to be completely phenyl red free for both cell cultures. The cells were cultivated over 3 days under these conditions before the compounds were tested.
- Route of administration:
- other: In-vitro study (not applicable)
- Positive control:
- As a positive control for the androgen receptor assay, methyltestosteron and flutamide were used.
- Details on results:
- Results: Spirodiclofen and Spirodiclofen-enol were not cytotoxic to either of the cell cultures. In the PALM cells Spirodiclofen and its metabolites had no androgenic or antiandrogenic activity up to a concentration of 10-5 M (Table 5.8.2.1.1.2g; Figure 5.8.2.1.1.2-4). In the receptor binding assay no binding was detected.
- Conclusions:
- A reporter gene assay using genetically engineered cells that express the human androgen receptor (PALM cells) did not reveal an androgenic or anti-androgenic potential of Spirodiclofen and its metabolites at concentrations of up to 10-5 M. In addition, no affinity for the androgen receptor could be demonstrated for Spirodiclofen and its metabolites in a receptor binding study. Thus, it can be excluded that receptor-mediated effects caused the testicular findings.
- Endpoint:
- mechanistic studies
- Remarks:
- Non-standard sub-acute study in the dog.
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- See main study report.
Referenceopen allclose all
Table 5.8.2.1.1.2g: Determination of the hormonal activity of Spirodiclofen, Spirodiclofen-enol, Hydroxy- Spirodiclofen-enol (Spirodiclofen-enol OH) in PALM cells at pH 7.2-7.4.
| Cells | Spirodiclofen | Spirodiclofen | Spirodiclofen-enol | Spirodiclofen-enol | Spirodiclofen-enol OH | Spirodiclofen-enol OH |
| NOEC (M) | EC50 (M) | NOEC (M) | EC50 (M) | NOEC (M) | EC50 (M) | |
| PALM | 10-5 | > 10-5 | 10-5 | > 10-5 | 10-5 | > 10-5 |
| PALM (+testos.) | 10-5 | > 10-5 | 10-5 | > 10-5 | 10-5 | > 10-5 |
> greater than
Description of key information
A number of studies are available which investigate the hormonal/ED effects of spirodiclofen.
Additional information
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