O,O,O-triphenyl phosphorothioate

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 Nov 2018 - 7 DEC 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: OECD 456

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: hormone system

Specific details on test material used for the study:

- Physical state/appearance: Solid/white
- Storage conditions: Room temperature, moisture protection

Dose / conc.:

0 other: µmol/l

Dose / conc.:

0 other: µmol/l

Dose / conc.:

0.001 other: µmol/l

Dose / conc.:

0.01 other: µmol/l

Dose / conc.:

0.1 other: µmol/l

Dose / conc.:

1 other: µmol/l

Dose / conc.:

10 other: µmol/l

Details on study design:

Final concentration of the test substance [µmol/L]: 0.00001, 0.0001, 0,001, 0.01, 1 and 10

Details on results:

The test article does influence the production of testosterone, based on the effects observed on relative testosterone and estradiol levels, and it is judged to be positive in the steroidogenesis assay using H295R cells.

For the evaluation of the relative increase/decrease in altered hormone production, the hormone values were normalized to the mean VC value of each the corresponding plate and results were expressed as changes relative to the VC in each test plate. The test substance statistically significantly decreased the testosterone levels at 1μM and 10 μM in experiment 1 and 2. It also statistically significantly increased the estradiol levels at 10μM in both experiments.

EXPERIMENT 1

					Forskolin		Prochloraz		Test substance
Concentration (µmol/L)			Untreated Control	Vehicle Control	1.00	10.00	0.10	1.00	0	0.00001	0.0001	0.00	0.01	0.10	1.00	10.00
Mitochondrial Activity	relative cell viability (%)	Mean	1.01	1.00	1.08	1.05	1.01	1.03	100.00	99.45	99.78	96.02	102.43	101.88	98.12	90.71
Testosterone	absolute (ng/mL)	Mean	1.20	1.10	1.93	2.00	0.40	0.12	0.94	0.90	0.93	0.94	0.91	0.89	0.74	0.33
	fold change	Mean	1.09	1.00	1.76	1.83	0.36	0.11	1.00	0.96	0.99	1.00	0.96	0.94	0.79*	0.35**
Estradlol	absolute (ng/ml)	Mean SD	0.01	0.01	0.56	0.58	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.04
	fold change	Mean SD	0.91	1.00	39.71	40.95	0.95	0.44	1.00	1.02	0.98	1.11	1.02	1.06	1.44	4.4**

Welch t-test (two sided): *p<=0.05; **p<=0.01

EXPERIMENT 2

					Forskolin		Prochloraz		Test substance
Concentration (µmol/L)			Untreated Control	Vehicle Control	1.00	10.00	0.10	1.00	0	0.00001	0.0001	0.00	0.01	0.10	1.00	10.00
Mitochondrial Activity	relative cell viability (%)	Mean	1.02	1.00	1.13	1.06	1.05	1.02	100.00	108.00	110.00	105.00	112.00	112.00	111.00	107.00
Testosterone	absolute (ng/mL)	Mean	1.37	1.22	2.00	2.00	0.55	0.15	1.09	1.12	1.04	1.06	1.06	1.03	0.92	0.44
	fold change	Mean	1.12	1.00	1.64	1.64	0.45	0.12	1.00	1.03	0.96	0.98	0.97	0.95	0.84*	0.41**
Estradlol	absolute (ng/ml)	Mean SD	0.01	0.01	0.51	0.64	0.02	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.04
	fold change	Mean SD	0.98	1.00	34.70	43.66	1.02	0.48	1.00	0.89	0.92	0.89	0.83	0.92	1.14	3.22**

Welch t-test (two sided): *p<=0.05; **p<=0.01

Conclusions:

In conclusion, under this experimental conditions, the test article does influence the production of testosterone, based on the effects observed on relative testosterone and estradiol levels, and it is judged to be positive in the steroidogenesis assay using H295R cells.

Executive summary:

The effects of the test item on cell viability and hormone levels are summarized in the table below. The direction of effects as increase (↑) or decrease (↓) is shown per hormone. Changes in hormone levels are only given for test substance concentrations that allow sufficient cell viability (equal to or above 80% relative to vehicle control treated cells).

Summary of effects of the test item on the steroidogenesis of H295R cells:

Effects observed (relative to vehicle control)	Experiment 1	Experiment 2	Max change	LOEC	NOEC
Cell viability	none	none	None	none	none
Testosterone	↓at 1-10 µmol/L	↓at 1-10 µmol/L	0.35-fold	1 µmol/L	0.1 µmol/L
Estradiol	↑at 10µmol/L only^a	↑at 10µmol/L only^a	4.40-fold	10 µmol/L^a	1 µmol/L^a

^aAs described in OECD TG 456 and OCSPP 890.1550, a test substance is to be judged to be positive if an effect at two adjacent concentrations in at least two independent experiments is observed.

LOEC: lowest observed effect concentration

NOEC: no observed effect concentration

In conclusion, under this experimental conditions, the test article does influence the production of testosterone, based on the effects observed on relative testosterone and estradiol levels, and it is judged to be positive in the steroidogenesis assay using H295R cells.

Reference 2

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2018-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 456

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes

Type of method:

in vitro

Endpoint addressed:

other: hormone system

Dose / conc.:

0 other: nM

Remarks:

Experiment 3

Dose / conc.:

0.001 other: nM

Remarks:

Experiment 3

Dose / conc.:

0.01 other: nM

Remarks:

experiment 1, 2 and 3

Dose / conc.:

0.1 other: nM

Remarks:

Experiment 1, 2 and 3

Dose / conc.:

1 other: nM

Remarks:

Experiment 1, 2 and 3

Dose / conc.:

10 other: nM

Remarks:

Experiment 1, 2 and 3

Dose / conc.:

100 other: nM

Remarks:

Experiment 1

Dose / conc.:

1 000 other: nM

Remarks:

Experiment 1, 2 and 3

Dose / conc.:

3 000 other: nM

Remarks:

Experiment 2

Dose / conc.:

10 000 other: nM

Remarks:

Experiment 1, 2

No. of animals per sex per dose:

in vitro

Control animals:

other: in vitro

Details on study design:

The final exposure concentrations were 10000 nM, 1000 nM, 100 nM, 10 nM, 1 nM, 0.1 nM and 0.01 nM in experiment 1, 10000 nM, 3000 nM 1000 nM, 10 nM, 1 nM, 0.1 nM and 0.01 nM in experiment 2 and 1000 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM and 0.0001 nM in experiment 3.

For details on study design, see "any other information on materials and methods incl. tables"

Details on results:

Results Steroidogenesis Assay Experiment 1
In steroidogenesis assay experiment 1, the tested concentrations were 0.01, 0.1, 1, 10, 100,
1000 and 10000 nM of O,O,O-Triphenyl phosphorothioate.
The mean basal estradiol production in the DMSO solvent control wells was 236 pg/mL. The
estradiol concentrations in medium from H295R cells exposed to the test item varied from
237 pg/mL to 285 pg/mL. The relative change of test item exposed wells compared to the
solvent treated control wells varied from 1.00 to 1.21. Statistical analysis showed that
O,O,O-Triphenyl phosphorothioate had no significant effect (p ≤ 0.05) on estradiol release in
H295R cells (see Appendix 5).
Since no statistically significant effect on estradiol release was observed, the results obtained
in the first experiment were considered negative.
The mean basal testosterone production in the DMSO solvent control wells was 4560 pg/mL.
The testosterone concentrations in the medium from the H295R cells exposed to the test item
varied from 2341 pg/mL to 6715 pg/mL. The relative change of test item exposed wells to
solvent treated control wells varied from 0.51 to 1.47. Statistical analysis showed that
O,O,O-Triphenyl phosphorothioate significantly (p ≤ 0.05) increased testosterone release at a
concentration of 0.01, 0.1, and 1 nM and significantly decreased testosterone release at a
concentration of 10000 nM in H295R cells (see Appendix 5).
The effects observed for testosterone at the low-end of the concentration curve (0.01, 0.1, and
1 nM) were very minor (fold-change <1.47-fold) and were therefore considered non-relevant
random effects e.g. caused by (biological) assay variations. However, the decrease in
testosterone concentration observed at 10000 nM could be a potential dose related effect, and
therefore the results for testosterone obtained in the first experiment were considered
equivocal.

Results Steroidogenesis Assay Experiment 2
To confirm the results obtained in experiment 1 and to better characterize the potential effect
on testosterone at the high end of the concentration curve, a test item concentration of
3000 nM was included in the second experiment. In steroidogenesis assay experiment 2, the
O,O,O-Triphenyl phosphorothioate concentrations tested were 0.01, 0.1, 1, 10, 1000, 3000
and 10000 nM.
The mean basal estradiol production in the DMSO solvent control wells was 118 pg/mL. The
estradiol concentrations in medium from H295R cells exposed to the test item varied from
138 pg/mL to 245 pg/mL. The relative change of test item exposed wells compared to the
solvent treated control wells varied from 1.17 to 2.08. Statistical analysis showed that
O,O,O-Triphenyl phosphorothioate significantly (p ≤ 0.05) increased estradiol release at a
concentration of 0.01, 0.1, 1 and 3000 nM in H295R cells (see Appendix 5).
For estradiol, the results were very variable. Since there was no clear dose response curve and
the effects observed at 0.01, 0.1, 1 and 3000 nM were not observed in experiment 1 and 3,
these effect were considered to be random effects e.g. caused by (biological) assay variations
and the results obtained for estradiol in experiment 2 were considered equivocal.
The mean basal testosterone production in the DMSO solvent control wells was 3418 pg/mL.
The testosterone concentrations in the medium from the H295R cells exposed to the test item
varied from 1656 pg/mL to 4660 pg/mL. The relative change of test item exposed wells to
solvent treated control wells varied from 0.48 to 1.36. Statistical analysis showed that
O,O,O-Triphenyl phosphorothioate significantly (p ≤ 0.05) increased testosterone release at a
concentration of 0.01, 0.1, and 1 nM and significantly decreased testosterone release at a
concentration of 10000 nM in H295R cells (see Appendix 5) and confirmed the results
obtained in experiment 1.
The effects observed for testosterone at the low-end of the concentration curve (0.01, 0.1, and
1 nM) were again very minor (fold-change <1.36-fold) and were therefore considered nonrelevant
random effects e.g. caused by (biological) assay variations. Again a decrease in
testosterone concentration was observed at 10000 nM. Therefore, the results for testosterone
obtained in the second experiment were considered equivocal.

Results Steroidogenesis Assay Experiment 3
Since in experiment 2 no effect on testosterone release was observed at 3000 nM, there was
no need to further investigate the effect at the high end of the concentration curve and the
third experiment was used to further investigate the effects at the low end of the concentration
curve. In steroidogenesis assay experiment 3, the O,O,O-Triphenyl phosphorothioate
concentrations tested were 0.0001, 0.001, 0.01, 0.1, 1, 10 and 1000 nM.
The mean basal estradiol production in the DMSO solvent control wells was 130 pg/mL. The
estradiol concentrations in medium from H295R cells exposed to the test item varied from
131 pg/mL to 185 pg/mL. The relative change of test item exposed wells compared to the
solvent treated control wells varied from 1.00 to 1.42. Statistical analysis showed that
O,O,O-Triphenyl phosphorothioate had a significant effect (p ≤ 0.05) on estradiol release at a
concentration of 0.001 nM in H295R cells (see Appendix 5).
The statistically significant effect on estradiol release observed at 0.001 nM was considered to
be a random effect, since the effect was not observed in experiment 1 and 2. Therefore, the
final conclusion for estradiol in experiment 3 was negative.
The mean basal testosterone production in the DMSO solvent control wells was 2329 pg/mL.
The testosterone concentrations in the medium from the H295R cells exposed to the test item
varied from 2257 pg/mL to 3325 pg/mL. The relative change of test item exposed wells to
solvent treated control wells varied from 0.97 to 1.43. Statistical analysis showed that
O,O,O-Triphenyl phosphorothioate significantly (p ≤ 0.05) increased testosterone release at a
concentration of 0.0001, 0.001, and 0.01 nM in H295R cells (see Appendix 5). However, a
statistical increase in testosterone release by 0.1 and 1 nM O,O,O-Triphenyl phosphorothioate
as observed in experiment 1 and 2 could not be repeated.
For testosterone, the results of experiment 3 did not confirm the results observed in
experiment 1 and 2. The effects observed for testosterone at the low-end of the concentration
curve were again very minor (fold-change <1.43-fold) and were therefore considered nonrelevant
random effects e.g. caused by (biological) assay variations. Therefore, the results for
testosterone obtained in the third experiment were considered negative.

Solubility Assessment

In the solubility test, O,O,O-Triphenyl phosphorothioate dissolved at a concentration of 1M

in DMSO. When this stock solution was diluted 1000-fold in exposure medium, precipitation

occurred. Since 10 mM was the highest stock concentration that produced a clear solution

after a 1000-fold dilution in medium, this concentration was used as highest exposure

concentration in the steroidogenesis assay experiments (final concentration in the well:

10 μM).

Chemical Hormone-Assay Interference Test

The results of the chemical hormone interference test are presented in Table 1 and Table 2

(Appendix 2). Compared to the basal hormone production, the interference of O,O,OTriphenyl

phosphorothioate (10 μM) with estradiol measurements was 8%. Since the

interference observed for testosterone (-25%) was more than the absolute value of 20%, the

experiment was repeated using 10 μM, 3 μM and 1 μM O,O,O-Triphenyl phosphorothioate.

In this experiment, the interference of O,O,O-Triphenyl phosphorothioate at 10 μM, 3 μM

and 1 μM was -9%, -8% and -11%, respectively. To confirm the latter result and to further

investigate the potential interference of the test item with the testosterone ELISA at lower

concentrations, a third experiment was performed. In this experiment, the interference of

O,O,O-Triphenyl phosphorothioate using 10 μM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM and

0.0001 nM was 2%, 2%, 5%, 72%, -4% and -2%, respectively. As 72% at 0.01 nM was more

than the absolute value of 20%, the experiment was repeated. In this experiment, the

interference of O,O,O-Triphenyl phosphorothioate using 0.1 nM, 0.01 nM and 0.001 nM was

-38%, -15%, and -13%, respectively.

Since the interference observed for the testosterone and estradiol hormone measurements was

below 20% for O,O,O-Triphenyl phosphorothioate at 10 μM, the interference of the test item

with the hormone assays was considered to be not significant.

Hormone Recovery Test

Medium samples spiked with 50 pg/mL and 250 pg/mL estradiol or 500 pg/mL and 2500

pg/mL testosterone were prepared and analyzed with ELISA to determine the recovery. The

results of the hormone recovery tests for estradiol and testosterone are presented in Table 3

and Table 4, respectively. The recovery of the estradiol spikes was 91%, 93%, 96% and

104%. The recovery of the testosterone spikes was 100%, 107%, 118% and 133%.

With the exception of 133%, the measured hormone concentrations did not deviate more than

30% of the nominal concentrations. Since the recovery of both 500 pg/mL testosterone

spiking solutions were both within the acceptance criteria (100% and 107%) while the

recovery of one of the 2500 pg/mL testosterone spiking solutions was also within the

acceptance criteria (118%), the ELISA assays were considered to be sufficiently accurate and

precise without cross-reactivity with components in the medium.

Exposure Experiments

Three steroidogenesis exposure experiments were performed. Exposure experiment 1 was

started on 15 May 2018, experiment 2 was started on 28 May 2018 and experiment 3 was

started at 11 June 2018. The data of experiment 1, 2 and 3 are discussed below.

Cell Viability Measurements

After each exposure experiment the viability of the H295R cells was determined using the

MTT test. Detailed results for the QC and test item plates are presented in Table 9 to

Table 14 (Appendix 2). The viability was expressed relative to the response in the solvent

controls, which was set at 100% viability (0% cytotoxicity). The test item did not affect cell

viability up to the highest test item concentration (10 μM).

Results QC plates and Assay Acceptability Criteria

To evaluate the performance of the assay a quality control (QC) plate was included in each of

the three steroidogenesis assay experiments. Estradiol and testosterone levels in the medium

samples from the QC and test item plates of experiment 1, 2 and 3 are presented in Table 15 -

Table 18, Table 19 - Table 22 and Table 23 - Table 26 (Appendix 2), respectively. A

comparison of the results obtained for the QC and test item plates with the acceptability

criteria for estradiol and testosterone is presented in the tables below.

For the estradiol measurements, all acceptability criteria were met with the exception of the

replicate measure CV for the SCs in experiment 2 on the test item plate. The high replicate

measure CV for the SCs (≤ 34%) were due to a few outliers, which were not seen in

experiment 1 and 3. In addition, inhibition of estradiol production using 1 μM prochloraz in

experiment 2 and 3 on the QC plate was 0.77-fold and 0.51-fold and therefore did not meet

the criteria of ≤ 0.5-fold-change compared to SC. In the same experiments, testosterone

production was inhibited 0.16-fold and 0.18-fold by 1 μM prochloraz and met the acceptance

criteria. Comparing the inhibitory effect of prochloraz between all experiments for estradiol

and testosterone demonstrated similar results. Taking this into account, it was concluded that

the results obtained for the QC and test item plates met the acceptability criteria in all three

experiments for both estradiol and testosterone.

The inter-plate CV for the SCs was 34% for estradiol and 21% for testosterone.

Effect of O,O,O-Triphenyl phosphorothioate on Estradiol and Testosterone Release in H295R Cells

The estradiol and testosterone levels in medium of H295R cells exposed to different

concentrations of O,O,O-Triphenyl phosphorothioate obtained in experiment 1, 2 and 3 are

presented in Table 17 - Table 18, Table 21 - Table 22 and Table 25 - Table 26 (Appendix 2),

respectively. The dose response curves for estradiol and testosterone obtained in experiment

1, 2 and 3 are presented in Figure 1 - Figure 6 in Appendix 1. The results of the statistical

analysis are presented in Appendix 5.

TextTable 5

Acceptability Criteria for Estradiol obtained inExperiment 1, 2 and 3

	Comparisonbetween	Estradiol
		Performancecriteria	Experiment1	Experiment2	Experiment3
QCplate
LOQ	NA	NA	10pg/mL	7 pg/mL	10pg/mL
Basal production ofhormoneintheSCs	Absoluteconcentration	NA	226pg/mL	109pg/mL	158pg/mL
	Fold-greaterthanLOQ	≥ 2.5-fold	22-fold	16-fold	15-fold
Within plate CV forSCs(replicatewells)	Absoluteconcentrations	≤30%	2.5%	7.3%	8.0%
Induction (10µM forskolin)	Fold-changecomparedtoSC	≥ 7.5-fold	20-fold	24-fold	21-fold
Inhibition (1µMprochloraz)	Fold-changecomparedtoSC	≤ 0.5-fold	0.39-fold	0.77-fold	0.51-fold
Hormone measurementsystem:Sensitivity	Detectable fold-changerelativetoSC	≥ 2.5-fold	22-fold	16-fold	15-fold
Hormone measurementsystem: ReplicatemeasureCVforSCs	Absoluteconcentrations	≤25%	≤13%	≤23%	≤14%
O,O,O-Triphenylphosphorothioate testitem plate
LOQ	NA	NA	10pg/mL	7 pg/mL	8 pg/mL
Basal production ofhormoneintheSCs	Absoluteconcentration	NA	236pg/mL	118pg/mL	130pg/mL
	Fold-greaterthanLOQ	≥ 2.5-fold	24-fold	18-fold	17-fold
Within plate CV forSCs(replicatewells)	Absoluteconcentrations	≤30%	9.9%	20%	20%
Hormone measurementsystem:Sensitivity	Detectable fold-changerelativetoSC	≥ 2.5-fold	24-fold	18-fold	17-fold
Hormone measurementsystem: ReplicatemeasureCVforSCs	Absoluteconcentrations	≤25%	≤14%	≤34%	≤ 7.2%

NA=Notapplicable,SC= Solventcontrol,CV= Coefficientofvariation,LOQ=LimitofQuantification.

TextTable 6

Acceptability Criteria for Testosterone obtained in Experiment 1, 2 and 3

	Comparisonbetween	Testosterone
		Performancecriteria	Experiment1	Experiment2	Experiment3
QCplate
LOQ	NA	NA	9 pg/mL	5 pg/mL	7 pg/mL
Basal production ofhormoneintheSCs	Absoluteconcentration	NA	3914 pg/mL	3486 pg/mL	3417 pg/mL
	Fold-greaterthanLOQ	≥ 5-fold	458-fold	697-fold	479-fold
Within plate CV forSCs(replicatewells)	Absoluteconcentrations	≤30%	9.2%	5.5%	9.5%
Induction (10µM forskolin)	Fold-changecomparedtoSC	≥ 1.5-fold	2.8-fold	2.9-fold	2.9-fold
Inhibition (1µMprochloraz)	Fold-changecomparedtoSC	≤ 0.5-fold	0.19-fold	0.16-fold	0.18-fold
Hormonemeasurementsystem: Sensitivity	Detectable fold-changerelativetoSC	≥ 5-fold	458-fold	697-fold	479-fold
Hormonemeasurement system:ReplicatemeasureCV forSCs	Absoluteconcentrations	≤25%	≤11%	≤20%	≤ 7.1%
O,O,O-Triphenylphosphorothioate testitem plate
LOQ	NA	NA	8 pg/mL	5 pg/mL	6 pg/mL
Basal production ofhormoneintheSCs	Absoluteconcentration	NA	4560 pg/mL	3418 pg/mL	2329 pg/mL
	Fold-greaterthanLOQ	≥ 5-fold	586-fold	626-fold	396-fold
Within plate CV forSCs(replicatewells)	Absoluteconcentrations	≤30%	15%	14%	3.8%
Hormonemeasurementsystem:Sensitivity	Detectable fold-changerelativetoSC	≥ 5-fold	586-fold	626-fold	396-fold
Hormonemeasurementsystem: ReplicatemeasureCVforSCs	Absoluteconcentrations	≤25%	≤24%	≤12%	≤16%

NA=Notapplicable,SC=Solventcontrol,CV=Coefficientofvariation,LOQ=LimitofQuantification

Conclusions:

In conclusion, all three H295R steroidogenesis assay experiments were valid. The test item O,O,O-Triphenyl phosphorothioate was considered to be negative for effects on estradiol release and equivocal for effects on testosterone release in the steroidogenesis assay. Overall the results for O,O,O-Triphenyl phosphorothioate obtained in the steroidogenesis assay were not interpretable.

Description of key information

OECD 456; non-GLP; NCI-H295Rcells; influences production of testosterone (2019)

OECD 456; non-GLP;NCI-H295Rcells; negative for effects on estradiol release and equivocal for effects on testosterone release in the steroidogenesis assay (2021).

Additional information

H295R assay (2019)

The test substance was investigated for its potential to influence the production of testosterone using the H295R Steroidogenesis Assay (according to OECD 456). Final concentrations analyzed of the test substance were 0.00001, 0.0001, 0,001, 0.01, 1 and 10 µmol/l.

For the evaluation of the relative increase/decrease in altered hormone production, the hormone values were normalized to the mean vehicle control (VC) value of each the corresponding plate and results were expressed as changes relative to the VC in each test plate. The test substance statistically significantly decreased the testosterone levels at 1 µM and 10 µM in experiment 1 and 2. It also statistically significantly increased the estradiol levels at 10 µM in both experiments. Maximal change for testosterone and estradiol was 0.35-fold and 4.40-fold, respectively. Changes in hormone levels are only given for test substance concentrations that allow sufficient cell viability (equal to or above 80% relative to vehicle control treated cells).

Under the experimental conditions chosen, the test substancedoes influence the production of testosterone, based on the effects observed on relative testosterone and estradiol levels, and it is judged to be positive in the steroidogenesis assay using H295R cells (2019).

H295R assay (2021)

In this study the potential of O,O,O-Triphenyl phosphorothioate to modulate the steroidogenic pathway, beginning with the sequence of reactions from cholesterol through the

production of testosterone and estradiol, was investigated using the human H295R cell line according to OECD Guideline 456 and GLP.

Three valid steroidogenesis assay experiments were performed whereby the test item was tested at seven concentrations up to 10 μM together with the positive control inducer forskolin and positive control inhibitor prochloraz. Dimethylsulphoxide (DMSO) was used as vehicle and the concentration of vehicle in the incubations was kept constant at 0.1% (v:v).

H295R cells were exposed for 48 hours to the vehicle, O,O,O-Triphenyl phosphorothioate and positive controls. After exposure, the viability of the cells was determined using the 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The concentration of estradiol and testosterone in the exposure medium was determined using commercially available Enzyme-Linked Immuno Sorbent Assays (ELISAs).

O,O,O-Triphenyl phosphorothioate did not affect cell viability up to a concentration of 10 μM. No clear interference of the test item with the estradiol and testosterone ELISAs was observed up to 10 μM, despite variable results.

With exception of the inhibition of estradiol production upon exposure of 1 μM prochloraz in experiment 2 and 3, all assay acceptability criteria i.e. the basal hormone production, within plate coefficient of variation (CV) and replicate measure CV for the DMSO solvent controls, hormone measurement sensitivity, induction of hormone production upon exposure of 10 μM forskolin exposure and inhibition of hormone production upon exposure of 1 μM prochloraz were met. Therefore, all steroidogenesis assay experiments were considered to be valid.

A summary of the results for O,O,O-Triphenyl phosphorothioate obtained in steroidogenesis experiment 1, 2 and 3 is presented in the table below.

Experiment No.	Effect on E2 Release	Decision	Effect on T Release	Decision
1	No effect	Negative	0.01 nM, 0.1 nM, 1 nM (increase) and 10000 nM(decrease)	Equivocal
2	0.01 nM, 0.1 nM, 1nM and 3000 nM(increase)	Equivocal	0.01 nM, 0.1 nM,1 nM (increase) and 10000 nM(decrease)	Equivocal
3	0.0001 nM (increase)	Negative^1)	0.0001 nM, 0.001 nM and 0.01 nM (increase)	Negative

1) Initial equivocal results were considered to be negative since the potential effect at 0.0001 nM was not observed in experiment 1 and 2.

In the first experiment, no statistically significant effect on estradiol release was observed, and therefore the results obtained in the first experiment were considered to be negative. In the second experiment a statistically significant increase in estradiol release was observed for 0.01, 0.1, 1 and 3000 nM test item. Since the results were highly variable, there was no clear dose response curve and the effects observed at 0.01, 0.1, 1 and 3000 nM were not observed in experiment 1 and 3, these effects were considered to be random effects and the final conclusion for estradiol in experiment 2 was equivocal. Since the results in experiment 3 were considered negative, overall, it can be concluded that O,O,O-Triphenyl phosphorothioate did not significantly alter estradiol release in H295R cells.

For testosterone release, the effects observed in experiment 1 and 2 at the low-end of the concentration curve were considered non-relevant random effects since the fold-changes were <1.5-fold, which is considered a relevant cut-off that was also used in the multi-laboratory validation report of the H295R steroidogenesis assay [2,3] and within the ToxCast program for screening of chemicals on effects on steroidogenesis using H295R cells [4]. However, the decrease in testosterone concentration observed at 10000 nM in experiment 1 and 2 could be a potential dose related effect, and therefore the results for testosterone were considered equivocal in experiment 1 and 2. Since in experiment 2 no effect at 3000 nM was observed, there was no need to further investigate the effect on testosterone release at the high end of the concentration curve. The third experiment was used to further investigate the effects on testosterone observed at the low end of the concentration curve. The results of experiment 3 did not confirm the results observed in experiment 1 and 2. The effects observed for testosterone at the low-end of the concentration curve were again very minor (fold-change <1.43-fold) and were therefore considered non-relevant random effects e.g. caused by (biological) assay variations. Therefore, the results for testosterone obtained in the third experiment were considered negative. Based on the outcome of experiment 1, 2 and 3 it can be concluded that the results obtained for O,O,O-Triphenyl phosphorothioate were considered equivocal.

Overall, the results obtained for the test item O,O,O-Triphenyl phosphorothioate in the steroidogenesis assay were considered, according to the criteria of the test guideline, not interpretable. The Lowest Observed Effect Concentration (LOEC) was 10000 nM (10 μM), which was the highest concentration tested.

In conclusion, all three H295R steroidogenesis assay experiments were valid. The test item O,O,O-Triphenyl phosphorothioate was considered to be negative for effects on estradiol release and equivocal for effects on testosterone release in the steroidogenesis assay. Overall the results for O,O,O-Triphenyl phosphorothioate obtained in the steroidogenesis assay were not interpretable.

Summary of steroidogenesis assays using H295R cells

 

In the two available steroidogenesis assays (2019 and 2021), the effect of the test substance on estradiol and testosterone production was assessed.

In the first assay (2019), two experiments were conducted at the same test substance concentrations of 0.01, 0.1, 1, 10, 100, 1000 and 10000 nM. In the second assay (2021), three experiments were conducted. The 1^stexperiment at test substance concentrations of 0.01, 0.1, 1, 10, 100, 1000 and 10000 nM, the 2^ndexperiment at test substance concentrations of 0.01, 0.1, 1, 10, 1000, 3000 and 10000 nM and the 3^rdexperiment at test substance concentrations of 0.0001, 0,001 0.01, 0.1, 1, 10 and 1000 nM.

 

When these five experiments are assessed together in a weight of evidenced approach evaluating concentrations where at least three experiments were performed, the overall interpretation is as follows:

 

Combined assessment based on the five experiments of the two steroidogenesis assays
Concentration [nM]		Number of experiments at this concentration		Estradiol (overall assessment based on at least 3 experiments)		Testosterone (overall assessment based on at least 3 experiments)
0,0001		1		Less than 3 experiments		Less than 3 experiments
0,001		1		Less than 3 experiments		Less than 3 experiments
0,01		5		No effect (in 4/5)		No effect (in 3/5)
0,1		5		No effect (in 4/5)		No effect (in 4/5)
1		5		No effect (in 4/5)		No effect (in 4/5)
10		5		No effect (in 5/5)		No effect (in 5/5)
100		3		No effect (in 3/3)		No effect (in 3/3)
1000		5		No effect (in 5/5)		No effect (in 3/5)
3000		1		Less than 3 experiments		Less than 3 experiments
10000		4		Equivocal (2/4 with increase)		Decreased(in 4/4)

 

As a clear result (decrease of testosterone production) was only observed at one concentration,it is concluded that the test substance does not influence the production of estradiol and only equivocally influences the production of testosterone.

YES/YAS assay

 

The test substance showedneither an estrogenic nor anti-estrogenic activityusing the YES-assay [ERa] (Yeast Estrogen Screening) (2012).

 

The test substance showedneither an androgenic nor anti-androgenic activityusing the YAS-assay [AR] (Yeast Androgen Screening) (2012.)