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Novo Nordisk

Filmdragerad tablett 1 mg/0,5 mg
(vit, rund, 6 mm, märkt NOVO 288)

Östrogen och gestagen, kombinationspreparat - systemisk effekt

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Miljöpåverkan (Läs mer om miljöpåverkan)

Estradiol

Miljörisk: Användning av estradiol har bedömts medföra medelhög risk för miljöpåverkan.
Nedbrytning: Estradiol bryts ned långsamt i miljön.
Bioackumulering: Estradiol har hög potential att bioackumuleras.


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Detaljerad miljöinformation

Environmental risk assessment of estrogens in pharmaceutical products marketed by Novo Nordisk in Sweden in 2016


17β-estradiol and its main metabolites estrone and estriol


Environmental risk: Use of 17β-estradiol has been considered to result in a moderate environmental risk. Both 17β-estradiol and its two main metabolites estrone and estriol are considered.


Degradation: 17β-estradiol is slowly degraded in the environment.


Bioaccumulation: 17β-estradiol is assessed not to have a high potential for bioaccumulation. The two main metabolites, estrone and estriol are considered to have a low poten-tial for bioaccumulation.


PBT/vPvB: Neither 17β-estradiol nor its two main metabolites are considered to be PBT/vPvB substances.


Detailed background information


1. The active pharmaceutical ingredients (API)


17β-estradiol is metabolized during human metabolism into the major transformation products estrone, estriol, estrone sulfate and estrone glucoronide (Ref. 28, 45, 60).


17β-estradiol, estrone and estriol are natural estrogens which belong to the class of steroid hormones. 17β-estradiol is the primary female sex hormone and estrone is the primary metabolite of 17β-estradiol. 17β-estradiol is used for hormone replacement therapy of women with menopause complications.


Chemical name 17β-estradiol (E2)

CAS no. 50-28-2

Molecular formula C18H24O2

Molecular weight 272.38 g/mol


Chemical name Estrone (E1)

CAS no. 53-16-7

Molecular formula C18H22O2

Molecular weight 270.37 g/mol


Chemical name Estriol (E3)

CAS no. 50-27-1

Molecular formula C18H24O3

Molecular weight 288.38 g/mol


2. Calculation of the risk quotient (PEC/PNEC)


2.1 Sold amount in Sweden

The total amount of estradiol (hemihydrate and valerat) sold in Sweden in 2016 was 38.0 kg API based on Quintiles IMS/LIF Health sales data.


2.2 Calculation of PEC in surface water

According to ref. 1, PEC (Predicted Environmental Concentration) in surface water is calculated according to the following formula:

PEC = 1.5*10-6*A*(100-R) µg/L, where


A = Total amount of API (kg) sold in Sweden in a given year. Reduction of A may be justified based on metabolism data. It can be assumed that 17β-estradiol is metabolised in the female body and excreted as 33% 17β-estradiol, 54% Estrone and 13% Estriol (ref. 5), so A is set to:

  • 17β-estradiol: 33% of 38.0 kg = 12.54 kg

  • Estrone: 54% of 38.0 kg = 20.52 kg

  • Estriol: 13% of 38.0 kg = 4.94 kg

R = Removal rate (%) due to loss by adsorption to sludge particles, by volatilization, hydrolysis or biodegradation. R = 0 if no data is available. The removal rates are based on estimation of distribution of estrogens in a municipal waste water treatment plant in accordance with the principles of the EU TGD and by use of the program SimpleTreat 3.0, which estimates the relative distribution of chemicals to each compartment: effluent, sludge and air. The following removal rates (R) in waste water treatment plants are estimated (ref. 5):

  • 17β-estradiol: 40% ; Conjugated 17β-estradiol: 6-8%. 17β-estradiol is excreted by mammals as glucuronide or sulfate conjugates in urine or in the unmetabolized form in feces. Adler et al. (ref. 9) reported that 50% of 17β-estradiol and 58% of estrone were conjugated in raw sewage. Furthermore, they found by measurement that 87% of the non-conjungated 17β-estradiol was removed in waste water treatment plant and 47% of the conjungated 17β-estradiol was removed. Overall, a measured removal of 67% was found for 17β-estradiol and its conjugates. Thus, it is considered conservative to keep the SimpleTreat estimated removal for 17β-estradiol of 40%.

  • Estrone: 8%; conjugated estrone: 0%. Adler et al. (ref. 9) measured that 55% of the estrone was removed whereas a slightly higher concentration of the conjugated in the effluent than in the effluent was found (approximately 7.5 ng/L conjugate in the inlet and 8 ng/L conjugate in the outlet). Overall, a measured removal of 19% was found for estrone and its conjugates. Thus, it is considered conservative to keep the SimpleTreat estimated removal for estrone of 8%.

  • Estriol: 2%; conjugates: 0%. Thus, an overall removal for estriol of 0% is assumed here.

On this basis the following PECs in surface water can be calculated:

  • PEC for 17β-estradiol: 1.5 * 10-6 * 12.54 * (100-40) = 0.0011 µg/L

  • PEC for estrone: 1.5 * 10-6 * 20.52 * (100-8) = 0.0028 µg/L

  • PEC for estriol: 1.5 * 10-6 * 4.94 * (100) = 0.00074 µg/L


2.3 Calculation of PNEC in surface water

Available eco-toxicological data for 17β-estradiol, estrone and estriol and the derivation of PNEC-values is presented in this section.


2.3.1 17β-estradiol

A proposed EQS (PNEC) value has been derived for the substance (ref. 7) in connection with setting 17β-estradiol on a short-list of 19 possible new priority substances for the Water Frame Directive (ref. 6). The data used for the derivation of the EQS-value is presented in Appendix together with the derivation, and only a short overview of the derivation is given here.


Knowledge of the mode of action of 17β-estradiol suggests that fish and amphibians are likely to be the most sensitive organisms. This is supported by the available chronic toxicity data which indicates that fish are particularly sensitive to 17β-estradiol. Two studies were located on amphibians with LOECs in the range of 1000-2740 ng/l reported for Rana pipens and Xenopus laevis. These LOECs are far above the NOECs for fish. Therefore, a SSD (Species Sensitivity Distribution) was derived for 17β-estradiol based on data for the most sensitive taxonomic groups, fish - expecting that chronic fish data used for the derivation of an SSD would also be protective of the other less sensitive group.


The lowest no observed effect concentration for 17β-estradiol is a 35-50 d NOEC of 0.5 ng/l (ref. 45) for the trout (Onchorhynchus mykiss). The observed effects were sperm volume, sperm density and fertilization success. The study was not carried out according to a guideline. Experiments took place in four identical flow-through 0.5 m3 tanks (three replicates and one control - each tank with 10 males and 3 females of approximate same size). Water inflow temperature were 6oC and air saturation of water was >90%. Fish were kept under natural photoperiod (experiments were carried out in Kreuzstein in Sankt Gilgen, Upper Austria during December – January).


Overall, reliable chronic NOEC values were available for 11 species of fish and the SSD was based on these 11 fish species (ref. 7). The HC5 for the SSD was found at 0.8 ng/l. Based on the available dataset and the knowledge of the mode of action an assessment factor of 2 was considered appropriate. This gives an AA-EQS of 0.4 ng/l.


This derivation of the AA-EQS was reviewed by SCHER (ref. 8). Both the reliability and the ecological relevance of the endpoints and taxonomic groups were considered. Overall, the SCHER supported the proposed AA-EQS of 0.4 ng/l for 17β-estradiol.


In conclusion, a PNEC of 0.4 ng/L is used for 17β-estradiol


2.3.2 Estrone

A PNEC-value has been derived for estrone in connection with setting the substance (together with 17β-estradiol) on a short-list of 19 possible new priority substances for the Water Frame Directive (ref. 6).

A well-accepted EU PNEC for estrone has been derived at 3.6 ng/l (ref. 56).

 

Environmental toxicity data for estrone has been collected and are presented in the annex.


As for 17β-estradiol, the mode of action for estrone suggests that fish and amphibians are likely to be the most sensitive organisms. Based on available data, fish is found to be the most sensitive species to estrone. A NOEC for estrone of 36 ng/l was obtained in 40-day study with Danio rerio (according to OECD Draft Test Guideline: A 40-day Juvenile Zebrafish Assay for screening of Endocrine Disrupting Chemicals), and a NOEC for estrone of 5 ng/l was obtained in a 90-day study (no guideline followed, fish specie: Oryzias latipes, effects measured: Organ weight in relationship to body weight; hatch, Vitellogenin 1 mRNA).


As for 17β-estradiol, the mode of action for estrone is well-known and fish is the most sensitive species. Therefore, an assessment factor of 10 for the chronic fish toxicity data is considered justified.


Using an assessment factor of 10, a PNEC of 0.5 ng/L was obtained.


2.3.3 Estriol

As for 17β-estradiol and estrone, the mode of action for estriole is well-known and fish is the most sensitive species. Therefore, an assessment factor of 10 for the chronic fish toxicity data is considered justified.


The No Observed Effect Concentration (NOEC) for induction of vitellogenin, which is considered a chronic eco-toxicity test, is found at 0.0465 µg/l for estriol (ref. 46; not-a guideline study; test species Oryzias latipes, duration of study 90 days, temperature: 25 ± 1 °C, three replicates and one control; 30 embryos per replicate).


Using an assessment factor of 10, a PNEC of 4.7 ng/L was obtained.


2.3.4 Derived PNECs

PNEC for the three APIs in surface water is:

  • PNEC for 17β-estradiol: 0.0004 µg/L

  • PNEC for estrone: 0.0005 µg/L

  • PNEC for estriol: 0.0047 µg/L


2.4 Calculation of the risk quotient (PEC/PNEC)

The following risk quotient PEC/PNEC can be calculated:

  • PEC/PNEC for 17β-estradiol: 0.0011/0.0004 = 2.75

  • PEC/PNEC for estrone: 0.0028/0.0005 = 5.6

  • PEC/PNEC for estriol: 0.00073/0.0047 = 0.15

The total risk quotient for 17β-estradiol, estrone and estriol is thus 8.5.


3. Information about degradation, bioaccumulation and PBT/vPvB


3.1. Degradation of 17β-estradiol

Activated sludge test according to OECD guideline no. 302A has shown that 17β-estradiol is inherently biodegradable under aerobic conditions in activated sludge (ref. 27). 17β-estradiol is thus slowly degraded in the environment. In a 100 days simulation study of 17β-estradiol (OECD Test Method no. 308), an aerobic mineralisation (marine) of 61±1% respectively 62±3% mineralisation (freswater) was found (ref. 78). Thus, 17β-estradiol is found to be biodegradable in both marine and fresh water. In addition, an activated sludge tests (OECD 302, ref. 2) show that 17β-estradiol is inherently biodegradable under aerobic conditions. Therefore, 17β-estradiol and its metabolites are assessed not to fulfil the criteria of persistence in the aquatic environment.


3.2. Bioaccumulation of 17β-estradiol, estrone and estriol

According to the FASS.se guidelines (ref. 1), substances with Log Pow ≥ 4 or BCF ≥ 500 are considered to have high potential for bioaccumulation. Valid BCF-data has prevalence above log Pow data. One limitation in the use of log Pow for the estimation of the bioaccumulation potential is that metabolism within the test organism is not considered.


The following data on bioaccumulation are retrieved from the literature and calculations:

Substance

Parameter

Result

Specie

Method

Reference

17β-estradiol (E2)

log Pow

3.94

n-octanol

Calculation

Ref. 75

17β-estradiol (E2)

BCF

38 (day 21); 43 (day 81); 45 (day 141)

High-back crucian carp (Carassius auratus)

No standard followed. 200 juvenile caged fish were exposed to waste water outlet at the secondary sedimentation tank (for up to 141 days). Concentrations in waste water and fish were measured.

Ref. 50

17β-estradiol (E2)

BCF

174

Male fathead minnow, plasma

Method: no standard followed. Male and female fathead minnow were to 17β-oestradiol for 19 days at nominal concentrations that ranged from 27.2-2740 ng l-1. Tissues were collected and the concentration in the plasma was measured. The estimated BCF was 174 in males based on the relationship between waterborne and plasma 17β-oestradiol

concentrations in surviving fish from all treatments.

Ref. 44

17β-estradiol (E2)

BCF

6.5

Larvae and juvenile flounder

Method: no standard followed. The estradiol uptake (through 48 hours) and depuration (through 48 hours) was studied both for larvae and juvenile flounders. Five test concentrations (between 4nM and 1000 nM) and a control was applied in the uptake study. No BCF could be established for females

Ref. 64

17β-estradiol (E2)

log Klip,w

Varied between 2.29 (vesicle including cholesterol)-3.79 (vesicle including unsaturated acyl chains).

Three types of synthetic membrane liposomes were tested.

Method: no standard followed. The partitioning between water and the synthetic membrane liposomes were measured by equilibrium dialysis

Ref. 79

Estrone (E1)

Log Pow

3.43

n-octanol

Calculation

Ref. 75

Estrone (E1)

BCF

35 (day 21); 29 (day 81); 35 (day 141)

High-back crucian carp (Carassius auratus)

No standard followed. 200 juvenile caged fish were exposed to waste water outlet at the secondary sedimentation tank (for up to 141 days). Concentrations in waste water and fish were measured.

Ref. 50

Estrone (E1)

BCF

241/278 (4hr), 229 (16 hr), 165 24 hr

Daphnia magna

No standard followed. Uptake of E1 by the D. magna. was measured at 4, 16, and 24 h and the final concentration of E1 in the pond water was analyzed by LC/MS at each time point. The experiment was repeated at a lower concentration of E1 (40mg/L) and uptake in the D. magna and concentration of E1 in the water was determined after 4 h. All bioconcentration experiments were carried out in triplicate.

Ref. 35


log Klip,w

Varied between 2.45 (vesicle including cholesterol)-3.92 (vesicle including unsaturated acyl chains).

Three types of synthetic membrane liposomes were tested.

Method: no standard followed. The partitioning between water and the synthetic membrane liposomes were measured by equilibrium dialysis

Ref. 79

Estriol (E3)

Log Pow

2.81

n-octanol

Calculation

Ref. 75

Estriol (E3)

log Klip,w

Varied between 0.179 (vesicle including cholesterol)-0.96 (vesicle including unsaturated acyl chains).

Three types of synthetic membrane liposomes were tested.

Method: no standard followed. The partitioning between water and the synthetic membrane liposomes were measured by equilibrium dialysis

Ref. 79


It is noted that 17β-estradiol has a calculated log Pow slightly below but close to the cut-off value of 4. It can be mentioned that a logPow slightly above 4 (4.01) has been measured (ref. 30, method not reported). Several measured BCFs are available for 17β-estradiol – all well below the cut-off value of 500. Therefore, 17β-estradiol is assessed not to have a high potential for bioaccumulation.


Both estrone and estriol have calculated log Pow well below 4. Actually, measured log Pow values are available for the two substances showing a log Pow of 3.13 respectively 2.45 (Ref. 30, method not reported). In addition, a BCF well below 100 is measured for estrone in the fish “high-back crucian carp”. Thus, both substances are considered to have a low potential for bioaccumulation.


Of some interest to note is the measured partitioning between water and synthetic membrane liposomes – mimicking biological specie-of the three substances. The partitioning of 17β-estradiol and estrone is on the very same level – whereas the partitioning of estriol to the membrane liposomes is much lower. This is in agreement with the calculated log Pow.


Overall, it is assessed that 17β-estradiol, estrone and estriol all have a low potential for bioaccumulation.


3.3. PBT/vPvB assessment

Persistence:

In a 100 days simulation study of 17β-estradiol (OECD Test Method no. 308) with a nominal concentrations 0.36 µg/L of unlabelled and 1.1 µg/L 14C-labelled E2, an aerobic mineralisation (marine) of 61±1% respectively 62±3% mineralisation (freswater) was found (ref. 78). Thus, 17β-estradiol is found to be biodegradable in both marine and fresh water. In addition, an activated sludge tests (OECD 302, ref. 2) show that 17β-estradiol is inherently biodegradable under aerobic conditions. Therefore, 17β-estradiol and its metabolites are assessed not to fulfil the criteria of persistence in the aquatic environment.


Bioaccumulation:

In addition, it was concluded in section 4.2 that neither 17β-estradiol nor its metabolites are assessed to have a high potential for bioaccumulation.


Environmental Toxicity:

According to ref. 1, the “T” criteria for toxicity is: ”Chronic NOEC < 0.01 µg/L”


The overall critical environmental toxicological effect from exposure to 17β-estradiol, estrone and estriol is the potential to affect population sustainability, e.g. reproductive output, hatching and fertilisation success. The induction of vitellogenin in fish – which may cause a change in sex from male to female - is an indicator of this effect.


The lowest effect concentration for 17β-estradiol is a 35-50 d NOEC of 0.5 ng/l (ref. 45) for the trout Onchorhynchus mykiss. This chronic NOEC for 17β-estradiol is significantly lower than the T-criteria. 17β-estradiol is thus regarded as toxic to aquatic organisms.


The NOEC for estrone for induction of vitellogenin and sex ratio for Danio rerio is measured at 36 ng/L, which is above the “T” criteria for toxicity.


The NOEC for estriol for induction of vitellogenin and sex ratio for Oryzias latipes is measured at 47 ng/L, which is above the “T” criteria for toxicity.


Conclusion regarding PBT/vPvB properties:

Considering all three aspects, 17β-estradiol, estrone and estriol do not meet the criteria for classification as a PBT or vPvB substance.


4. Environmental risk classification of estrogens


In conclusion:

  • The total risk quotient for 17β-estradiol, estrone and estriol is 8.5.

  • 17β-estradiol, estrone and estriol do not meet the criteria for classification as a PBT or vPvB substance.


Based on the calculated risk quotients and information about degradation, bioaccumulation and eco-toxicity of 17β-estradiol, estrone and estriol the following environmental risk phrase should be applied to pharmaceutical products with estrogens according to the criteria in the FASS.se guidelines (ref. 1):


”Use of pharmaceutical products with estrogens has been considered to result in moderate environmental risk”


This risk phrase is according to the FASS.se guidelines applicable for risk quotients in the interval: 1 < PEC/PNEC ≤ 10.



5. References

General references

1. Environmental classification of pharmaceuticals at FASS – Guidance for pharmaceutical companies 2012.

2. D'Ascenzo G., A. Di Corcia, A. Gentili, R. Mancini, R. Mastropasqua, M. Nazzari, et al. Fate of natural estrogen conjugates in municipal sewage transport and treatment facilities. Sci. Total Environ, 301 (2003), pp. 199-209

3. DHI (2001): Litteratur-review over økotoksikologiske data for østradiol og østron. November 2001. Udført af DHI. (only in Danish)

4. DHI (2003): Summary of selected investigations performed for Novo Nordisk A/S - Steroid hormones. October 2003. Prepared by DHI.

5. DHI (2003): Fate and effects of humanly excreted estrogens - 17 β-estradiol, estrone, estriol and ethinylestradiol. October 2003. Prepared by DHI.

6. European Union (2013). “Directive 2013/39/EU of the European Parliament and of the Council of 12 August 2013 amending Directives 2000/60/EC and 2008/105/EC as regards priority substances in the field of water policy”.

7. EU (2011): Beta-estradiol EQS dossier 2011.

8. SCHER (Scientific Committee on Health and Environmental Risks) (2011). OPINION ON "CHEMICALS AND THE WATER FRAMEWORK DIRECTIVE: DRAFT ENVIRONMENTAL QUALITY STANDARDS" 17β-estradiol (E2) SCHER adopted this opinion at its 12th plenary on 30 March 2011. 

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52. Metcalfe C D, Metcalfe T L, Kiparissis Y, Koenig B, Khan C, Hughes R J, Croley T R, March R E , Thomas P. (2001). Estrogenic potency of chemicals detected in sewage treatment plant effluents as determined by in vivo assays with Japanese medaka (Oryzias latipes). Environmental Toxicology and Chemistry 20(2): 297-308.

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Appendix


Nitrification inhibition test with activated sludge:

Substance

Method

Concentration & Exposure time

Effect parameter

EC20

Reference

17β-estradiol

ISO 9509

62,5–1.000 µg/L

2 hrs

Inhibition of nitrification rate

> 918 µg/L

Ref. 23

Estrone

ISO 9509

62,5–1.000 µg/L

2 hrs

Inhibition of nitrification rate

> 172 µg/L

Ref. 24


The studies did not show significant inhibition of the nitrification rate in activated sludge at the tested concentrations.


Biodegradation test of 17β-estradiol:

Substance

Method

Concentration & Exposure time

Result

Reference

17β-estradiol (E2)

OECD Test Method no. 308: “Aerobic transformation of 17β-estradiol in aquatic sediment systems”

Nominal concentrations 0.36 µg/L and 1.1 µg/L of unlabelled and 14C-labelled E2, respectively

100 days

61±1% mineralisation (marine)

62±3% mineralisation (freswater)

Ref. 78

17β-estradiol

OECD Test Method no. 301D: “Closed Bottle Test”

1.64 mg/L

28 days

3.5-9.8 % of ThoD

Ref. 26

17β-estradiol (E2)

OECD Guideline no. 302A: “Inherent Biodegradability: Modified SCAS Test” and “Activated Sludge Biodegradability Simulation Test”

Ca. 20 µg/L

Aerobic: 48 hrs

Anoxic: 8 days

Aerobic:

See below *

Anoxic:

No significant degradation

Ref. 27


* Results according to OECD Guideline no. 302A:

  • The total 14C-concentration decreased by 70% of the initial added 14C within the first 45 minutes of the test period

  • During the first 45 minutes of the test period, a 1. order rate constant was estimated at 2.2 ± 0.2 L*day-1*gSS-1 for the total test substance concentrations > 2.5 µg E2/L

  • During the test period from 3-48 hours, a 1. order rate constant was estimated at 0.031 ± 0.003 L*day-1*gSS-1 for the total test substance concentrations < 2.5 µg E2/L


On basis of the biodegradation test results it can be concluded that:

  • 17 β-estradiol is not readily degradable under closed bottle conditions since the minimum requirement BOD = 60% of ThOD within 10 days is not fulfilled.

  • 17 β-estradiol is inherently biodegradable under aerobic conditions but not under anoxic conditions in activated sludge simulation.


Reproduction test for 17β-estradiol on the earth worm, Enchytraeus albidus

Method

Concentration &

Exposure time

Effect parameter

NOEC

Reference

OECD Draft Test Guideline 220: “Enchytraeidae Reproduction Test”, March 2000 and in agreement with the existing OECD Guideline No. 220: Enchytraeid Reproduction Test

50–1,000 mg/kg soil d.w.

21 days

Adult mortality

Inhibition of reproduction

Changes in behaviour and/or morphology

> 1,000 mg/kg

Ref. 25

The study did not show significant effect on neither of the stated parameters at the tested concentrations.


Derivation of PNEC for 17β-estradiol

A suggestion for AA-EQS has been drafted and reviewed (ref. 7). The below derivation is based on this derivation.

Specie Group

Organism

Effect

Duration

End-Point

Value (µg/L)

KLIMISH Score

Reference

Short Term Data

Algae

Desmodesmus subspicatus

Growth (GLP)

72 h

EC50

>3100

1

Ref. 61

Invertebrate

Acartia tonsa

Mortality

48 h

EC50

>1000

2

Ref. 10

Fish

Cyprinus carpio

VTG induction in hepatocytes

3 d

EC50

24.52

2

Ref. 62

Fish

Oncorhynchus mykiss

Mortality

96 h

LC50

>500

1

Ref. 60

Fish

Oncorhynchus mykiss

VTG induction in hepatocytes

3 d

EC50

7.08

2

Ref. 62

Fish

Oryzias latipes

Egg and embryo mortality

72 h

LC50

460

2

Ref. 41

Fish

Oryzias latipes

Adult

72 h

LC50

3500

2

Ref. 41

Long-term data

Algae

Desmodesmus subspicatus

Growth

72 h

NOEC

>3100

1

Ref. 61

Algae

Pseudokirchneriella subcapitata

Growth (OECD 201, GLP)

72 h

NOEC

>523

2

Ref. 77

Arthropoda

Balanus amphrite

larval colonization

2 d

NOEC

=0.1

2

Ref. 11

Invertebrate

Acartia tonsa

development

5 d

EC10

370

2

Ref. 10

Invertebrate

Acartia tonsa

development

5 d

EC50

720

2

Ref. 10

Invertebrate

Acartia tonsa

Reproduction

GLP, Not a guideline study;

21 d

NOEC

>368

2

Ref. 13

Invertebrate

Ceriodaphnia dubia

reproduction

7 d

NOEC

=10000

2

Ref. 70

Copepoda

Nitocra spinipes

reproduction

18 d

NOEC

≥160

2

Ref. 14

Copepoda

Tisbe battagliai

reproduction

21 d

NOEC

≥100

2

Ref. 34

Amphibien

Xenopus laevis

feminization

84 d

LOEC

2.74

2

Ref. 42

Amphibien

Rana pipiens

Intersex

162 d

LOEC

≤1

2

Ref. 51

Fish

Cyprinodon variegatus

Proportion of viable eggs F1 and F2

280 d

LOEC

0.04

2

Ref. 16

Fish

Cyprinodon variegatus

Proportion of viable eggs F1 and F2

280 d

NOEC

0.01

2

Ref. 16

Fish

Danio rerio

altered gonadal histology, sex ratio

21 d

LOEC

0.1

2

Ref. 15

Fish

Danio rerio

altered gonadal histology, sex ratio

21 d

NOEC

0.025

2

Ref. 15

Fish

Danio rerio

altered gonadal histology, secondary sexual characteristics

21 d

NOEC

0.005

2

Ref. 15

Fish

Danio rerio

reproduction

200 d

NOEC

≤0.005

2

Ref. 53

Fish

Danio rerio

Egg number in the clutch and hatching

21 d

NOEC

0.087

2

Ref. 66

Fish

Gabiocypris rarus

sex ratio

21 d

LOEC

0.025

2

Ref. 48

Fish

Gabiocypris rarus

sex ratio

21 d

NOEC

0.005

2

Ref. 48

Fish

Gambusia holbrooki

reproductive success

84 d

LOEC

0.02

2

Ref. 28

Fish

Gambusia holbrooki

reproductive success

84 d

NOEC

0.1

2

Ref. 28

Fish

Melanotaenia fluviatilis

egg production

14 d

LOEC

0.3

2

Ref. 58

Fish

Melanotaenia fluviatilis

egg production

14 d

NOEC

0.1

2

Ref. 58

Fish

Oncorhynchus mykiss

Sperm volume, sperm density and fertilization success

35-50 d

LOEC

0.001

2

Ref. 45

Fish

Oncorhynchus mykiss

Sperm volume, sperm density and fertilization success

35-50 d

NOEC

0.0005

2

Ref. 45

Fish

Oryzias javanicus

Fertility of the eggs

187 d

LOEC

0.016

2

Ref. 37

Fish

Oryzias javanicus

Fertility of the eggs

187 d

NOEC

0.0095

2

Ref. 37

Fish

Oryzias latipes

Gender shift (testis-ova)

90 d

LOEC

0.1

2

Ref. 52

Fish

Oryzias latipes

Gender shift (testis-ova)

90 d

NOEC

0.01

2

Ref. 52

Fish

Oryzias latipes

total study

90 d

LOEC

0.004

3

Ref. 52

Fish

Oryzias latipes

total study

90 d

NOEC

0.0004

3

Ref. 52

Fish

Oryzias latipes

feminization

200-300 d

NOEC

0.1

2

Ref. 69

Fish

Oryzias latipes

reduced fertility

59 d

NOEC

0.0029

2

Ref. 66

Fish

Oryzias latipes

feminization

28 d

LOEC

≤0.01

2

Ref. 54

Fish

Oryzias latipes

number of eggs

14 d

NOEC

0.272

2

Ref. 68

Fish

Oryzias latipes

reduced fertility

21 d

NOEC

0.227

2

Ref. 40

Fish

Oryzias latipes

Hatching time

20 d

NOEC

0.034

2

Ref. 29

Fish

Oryzias latipes

various reproduction endpoints

14 d

NOEC

0.379

3

Ref. 39

Fish

Pimephales promelas

Feminization and weight gain

91 d

LOEC

0.0279

1

Ref. 60

Fish

Pimephales promelas

Feminization and weight gain

91 d

NOEC

>0.008

1

Ref. 60

Fish

Pimephales promelas

reduced egg production

19 d

EC10

0.0066

2

Ref. 43

Fish

Pimephales promelas

reproduction, reduced egg production

21 d

NOEC

0.044

3

Ref. 78

Fish

Poecilia reticulata

Feminization (GSI, sex ratio)

90 d

LOEC

0.5

2

Ref. 74

Fish

Poecilia reticulata

Feminization (GSI, sex ratio)

90 d

NOEC

0.1

2

Ref. 74

Fish

Pomatoschistus minutus

reproduction

240 d

NOEC

0.097

2

Ref. 59

Fish

Thymallus thymallus

Sperm volume, motility of sperm

50 d

LOEC

≥0.001

2

Ref. 45


Acute effects have been considered of no relevance and therefore no MAC-EQS has been derived.


Chronic toxicity data for 17β-estradiol is available for a range of species including algae, crustaceans, rotifers, amphibians and fish. It is concluded that the critical effect due to exposure of 17β-estradiol and its primary metabolites estrone and estriol is the induction of vitellogenin in fish that may cause a change in sex from male to female.


In order to apply the SSD (Species Sensitivity Distribution) approach the available dataset should preferably contain more than 15, but at least 10 NOECs/EC10s from different species covering at least 8 taxonomic groups. For estimating an AA-EQS freshwater using the SSD approach the following taxa would normally need to be represented, i.e.

  • a fish species

  • a second family in the phylum Chordata

  • a crustacean

  • an insect

  • a family in a phylum other than Arthropoda or Chordata

  • a family in any order of insect or any phylum not represented

  • algae

  • a higher plant

The available chronic toxicity dataset for 17β-estradiol does not meet the data requirements for using the SSD approach. However, 17β-estradiol is a naturally occurring hormone and has a specific mode of action with effects on the reproductive physiology of vertebrates. The EU guidance notes that if a chemical is known to have a specific mode of action an SSD can be derived for only those taxa that are expected to be particularly sensitive.


Knowledge of the mode of action of 17β-estradiol suggests that fish and amphibians are likely to be the most sensitive organisms. This is supported by the available chronic toxicity data which indicates that fish are particularly sensitive to 17β-estradiol. Two studies were located on amphibians with LOECs in the range of 1000-2740ng/l reported for Rana pipens and Xenopus laevis. It is therefore proposed that an SSD is derived for β -estradiol based on data for the most sensitive taxonomic groups. It is expected that based on knowledge of the mode of action the chronic fish data the derivation of an SSD based on fish species only should be protective of other less sensitive group.


Reliable chronic NOEC values were available for 11 species of fish. An SSD has therefore been derived based on 11 fish species. For several species a number of different studies have been reported. The EU guidance on the derivation of an SSD indicates that where a number of data points are available for a species a geometric mean should be calculated to propose a single value for a species. This approach is not appropriate for all the available data as the studies are often non-standard and consider a range of endpoints and exposure durations and are therefore not directly comparable. In these cases, the lowest NOEC value is used for a species.


The SSD based on the fish data is shown below. The distribution fit to a log normal distribution.

Affected species

The HC5 from the above SSD is 0.8 ng/l. An assessment factor in the range of 1-5 should be applied to the HC5 based on the guidance given in the TGD-EQS (E.C., 2011). Based on the available dataset and the knowledge of the mode of action it is considered that an assessment factor of 2 (mode of toxic action is well understood, HC5 has been derived based on data for the most sensitive taxonomic group, a wide range of endpoints and durations including population relevant endpoints such as hatching, fertilisation, changes in sex ratio are included in the dataset) is appropriate for the derivation of the AA-EQS.

This gives a EQS of 0.4 ng/l.


The derivation of the AA-EQS has been reviewed by SCHER (ref. 8). Both the reliability and the ecological relevance of the endpoints and taxonomic groups have been considered. Overall, the SCHER supports the proposed AA-EQS of 0.4 ng/l.


Derivation of PNEC for estrone

Specie Group

Organism

Effect

Duration

End-Point

Value (µg/L)

KLIMISH Score

Reference

Short Term Data

Algae

Pseudokirchneriella subcapitata

Growth (OECD 201)

72 h

EC50

>451

1

Ref. 66

Crustacean

Acartia tonsa

Mortality

48 h

NOEC

≥1000

2

Ref. 10

Crustacean

Neomysis integer

Mortality

96 h

LC50

>10000


Ref. 18

Copepoda

Tisbe battagliai

Mortality

10 d

LC50

≥100


Ref. 28

Echinoderm

Strongylocentrotus purpuratus

Development

96 h

EC50

6,4.4

2

Ref. 60

Long-term data

Algae

Pseudokirchneriella subcapitata

Growth (OECD 201)

72 h

NOEC

≥451

2

Ref. 66

Crustacean

Acartia tonsa

Development

5 d

EC10

250

2

Ref. 10

Copepoda

Tisbe battagliai

Sex ratio; Re-production (method #1)

21 d

NOEC

≥100

2

Ref. 28

Fish

Danio rerio

Vitellogenin induction, sex ratio (OECD Draft Test Guideline: A 40-day Juvenile Zebrafish Assay for screening of Endocrine Disrupting Chemicals)

40 d

NOEC

0.036

2

Ref. 22

Fish

Danio rerio

Vitellogenin 1 mRNA; XPA mRNA; XPC mRNA

4 d

NOEC

0.1


Ref. 55

Fish

Danio rerio

Ovarian Somatic Index (OSI)

21 d

EC10

0.195

2

Ref. 77

Fish

Danio rerio

Vitellogenin induction

21 d

EC10

0.139

2

Ref. 77

Fish

Oncorhynchus mykiss

VTG-Induction (adult)

21 d

NOEC

0.048

2

Ref. 61

Fish

Oncorhynchus mykiss

VTG-Induction (adult)

14 d

NOEC

0.0032

3

Ref. 74

Fish

Oryzias latipes

Feminization


NOEC

0.1


Ref. 52

Fish

Oryzias latipes

Imposex, intersex conditions

- d

NOEC

<0.008


Ref. 52

Fish

Oryzias latipes

Hatch

15 d

NOEC

0.005


Ref. 46

Fish

Oryzias latipes

Vitellogenin 1 mRNA

90 d

NOEC

0.005


Ref. 46

Fish

Oryzias javanicus

Time to hatch


NOEC

0.198


Ref. 38

Fish

Oryzias javanicus

Number of eggs; number of fertilized eggs, time to hatch

239 d

NOEC

0.484


Ref. 38

Fish

Pimephales promelas

Vitellogenin induction (method #2)

21 d

NOEC

0.01

2

Ref. 57

Fish

Pimephales promelas

Egg production


NOEC

0.098


Ref. 73

Fish

Pimephales promelas

Hatch

4 d

NOEC

0.781


Ref. 73

Fish

Pimephales promelas

Organ weight in relationship to body weight; Sexual development; stage; Vacuolization

21 d

NOEC

0.054


Ref. 17

Fish

Pimephales promelas

Vitellogenin

4 d

NOEC

0.034


Ref. 73

Fish

Pimephales promelas

Vitellogenin

21 d

NOEC

0.054


Ref. 17

Fish

Pimephales promelas

Number of eggs

21 d

NOEC

0.307


Ref. 73

Fish

Pimephales promelas

Plasma vitellogenin

21 d

NOEC

0.00074


Ref. 74

Fish

Salmo trutta

Vitellogenin

10 d

NOEC

0.063


Ref. 18


Method#1: Newly released 24 h old species were exposed to the substance dissolved in sea water. Effects monitored in terms of survival, development and sex ratio after 10 days at 20oC. Adult males and females were then paired and exposures continued to investigate effects on reproductive output after 21 days total exposure.

Method#2: The effects on the plasma vitellogenin level and gonadosomatic index of male fathead minnows (Pimephales promelas) was studied in a continuous flow exposure system for 21 days. All fish were acclimated to the test conditions for a period of 24 h before the start of the exposure.


Derivation of PNEC for estriol

Specie Group

Organism

Effect

Duration

End-Point

Value (µg/L)

KLIMISH Score

Reference

Short Term Data

-

-







Long-term data

Fish

Danio rerio

Vitellogenin (method#1)

18 d

NOEC

0.3


Ref. 32

Fish

Danio rerio

Survival (method#1)

40 d

NOEC

21.7


Ref. 32

Fish

Danio rerio

Sex ratio (method#1)

40 d

NOEC

6.7


Ref. 32

Fish

Oryzias latipes

Abnormal(method#2)

15 d

NOEC

0.4622


Ref. 46

Fish

Oryzias latipes

Hatch

(method#2)

15 d

NOEC

0.04651


Ref. 46

Fish

Oryzias latipes

Sex ratio

(method#2)

30 d

NOEC

4.517


Ref. 46

Fish

Oryzias latipes

Vitellogenin 1 mRNA; hatch; Organ weight in relationship to body weight

(method#2)

90 d

NOEC

0.04651


Ref. 46

Fish


Oryzias latipes

Estrogen receptor alpha mRNA; Organ weight in relationship to body weight

(method#2)

90 d

NOEC

4.517


Ref. 46

[1]It was found that the Vtg gene in male medaka fish can be induced by estriol at environmentally relevant concentration of 5 ng/L. However, it was noted that the Vtg mRNA changes are hardly ever reflected in concomitant changes in functional protein. Therefore, further studies were concluded to be needed to detect more sex hormone pathway gene expressions and functional protein levels to evaluate comprehensively estrogen potency of estriol in fish.


Method#1: A Fish Sexual Development Test (FSDT) (an extension of the existing OECD TG 210, fish early life stage toxicity test).

Method#2: Measurement of the impact of estriol on the embryonic development, sex differentiation, growth, and changes of functional genes related to reproduction of medaka (O. latipes) exposed to different concentrations of estriol during embryo-larval-, juvenile- and adult life stages. The corresponding time to hatching, hatchability, gross abnormalities, sex ratio, hepatosomatic index (HSI), gonadosomatic index (GSI), and changes of Vtg-I and ERα genes in livers of the fish exposed to estriol for 90 days were determined. Embryos less than 4 h post-fertilization were used in the exposure experiments. The embryos were exposed to nominal estriol concentrations of 5, 50, 500 and 5000 ng/L in charcoal-dechlorinated tap water for 15 days. Each exposure level had 3 replicate test concentrations with 30 embryos per replicate. In addition, solvent controls (SC) were included in the experimental design. The embryos in each group were placed in a glass dish and incubated on a 16:8 h light: dark photoperiod cycle at 25 ± 1 °C. Eighty percent of the test solution was renewed every 24 h. Hatchability, time to hatching and gross abnormalities were recorded. Once hatched, the hatched fry were continuously maintained at the same concentrations for the additional 15 days. After the additional 15 days of exposure, the genetic sex ratio was determined. Ten fish including five females and five males were assigned randomly to a 5-L glass aquarium and duplicate aquaria were used at each exposure level. Fish were continuously exposed to nominal estriol concentrations of 5, 50, 500, and 5000 ng/L and the SC was included in the experiment design. The solution was renewed every 24 h. Treated and control fish were exposed for another 60 days. The entire test duration was 90 days.


Noretisteron

Miljörisk: Risk för miljöpåverkan av noretisteron kan inte uteslutas då det inte finns tillräckliga ekotoxikologiska data.
Nedbrytning: Noretisteron är potentiellt persistent.
Bioackumulering: Noretisteron har låg potential att bioackumuleras.


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Detaljerad miljöinformation

Environmental risk assessment of norethisterone acetate (NETA) in pharmaceutical products marketed in Sweden in 2016


This document includes environmental risk assessment of norethisterone acetate (NETA) in pharmaceutical products marketed in Sweden in 2016. The risk assessment is performed in accordance with the FASS.se guidelines on environmental classification of pharmaceuticals (ref. 1).


1. The active pharmaceutical ingredient


Norethisterone acetate (NETA), also known as norethindrone acetate, is a steroidal progestin that is used as a hormonal contraceptive. It is an acetate ester of norethisterone which belongs to the class of steroid hormones.


Chemical name Norethisterone Acetate (NETA)

CAS no. 51-98-9

Molecular formula C22H28O3

Molecular weight 340.46 g/mol

Water solubility 4.4 mg/L at 20ºC


2. Eco-toxicological data


The following tests have been performed with norethisterone or norethisterone acetate (NETA):

• Immobilisation test on dapnia

• Growth inhibition test on green alga

• Growth inhibition test on bacteria

• Biodegradation

• Octanol/water coefficient


The tests are performed by Schering in accordance with OECD principles for good laboratory practice.


Immobilisation test on dapnia (ref 2 and 3):

Acute immobilization test with norethisterone and NETA on Daphnia magna, 48h:

• No effect was observed at saturated concentration. EC50 > 4.4 - 4.6 mg/L.

Method: OECD guidelines for testing of chemicals, no. 202: “Daphnia sp., Acute immobilisation test and reproduction test”.


Growth inhibition test on green alga (ref 4):

Growth inhibition test with NETA on the green alga Desmodesmus subspicatus:

• EC50 (biomass): 0.4 mg/L

• EC50 (growth rate): 0.6 mg/L


Method: OECD guidelines for testing of chemicals, no. 201: “Alga, growth inhibition test”


Growth inhibition test on bacteria (ref. 5):

Growth inhibition test with norethisterone on the bacterium Pseudomonas putida:

• No inhibition at saturated concentration (ca. 7.8 mg/l)


Method: Schering method no. TX.ME.572.3 and DIN 38412 L8, March 1991.


Biodegradation test (ref. 6 and 7):

Biodegradation test on norethisterone and NETA:

• norethisterone and NETA are not readily biodegradable under “modified Sturm test” conditions since less than 10% of the substance was biodegraded within 28 days.


Method: OECD guideline for testing of chemicals, Ready biodegradability: CO2-evolution test, no 301B.


Octanol/water coefficient (ref. 8) Method Unknown:

The octanol/water coefficient for NETA has been determined to LogPow = 3.7. Since LogPow < 4 it indicates that NETA has low potential for bioaccumulation according to ref. 1.


Summary of test results:

The obtained test results indicate that norethisterone/NETA:


• is very toxic to the green alga Desmodesmus subspicatus (since EC50 < 1 mg/L)

• has no effect on immobilisation of Daphnia magna

• has no effect on the growth of the bacteria Pseudomonas putida

• is not readily biodegradable

• has low potential for bioaccumulation


3. Calculation of the risk quotient (PEC/PNEC)


3.1. Sold amount in Sweden

The total amount of norethisterone and norethisterone acetate (NETA) sold in Sweden in 2016 was 14.90 kg API based on QuintilesIMS/LIF Health sales data.


3.2. Calculation of PEC in surface water

According to ref. 1, PEC (Predicted Environmental Concentration) in surface water is calculated according to the following formula:


PEC = 1.5*10-6*A*(100-R) µg/L, where


• A = Total amount of API (kg) sold in Sweden in a given year. Reduction of A may be justified based on metabolism data.

• R = Removal rate (%) due to loss by adsorption to sludge particles, by volatilization, hydrolysis or biodegradation. R = 0 if no data is available.


Due to lack of data, the calculation of PEC of NETA in surface water is based on the following assumptions:


• no metabolism of in the body

• no removal in waste water treatment plants.


Without taking removal effects of metabolism and waste water treatment into consideration the following PEC of NETA in surface water can be calculated according to ref. 1:


• PEC = 1.5*10-6*14.90*(100-0) = 0.00224 µg/L.


Since PECSurface water is below the action limit 0.01 µg/L stated in ref. 1 it is unlikely that NETA constitutes a significant risk for the environment.


3.3. Calculation of PNEC in surface water

According to ref. 1, calculation of PNEC (Predicted No Effect Concentration) in surface water should be based on eco-toxicological data for three trophic levels. However, it has only been possible to present eco-toxicological data for two trophic levels i.e. green alga and daphnia. Furthermore, it is not known if these organisms are the most sensitive to NETA.


Consequently, it is not possible to calculate a valid PNEC according to the requirement in ref. 1 on basis of the available eco-toxicological data.


3.4. Calculation of the risk quotient (PEC/PNEC)

The risk quotient (PEC/PNEC) cannot be calculated for the reason stated in section 3.3.


4. PBT and vPvB assessment


Persistence:

NETA is not readily biodegradable under “modified Sturm test” conditions since less than 10% of the substance was biodegraded within 28 days (ref. 6 and 7).


According to ref. 1, it cannot be excluded that NETA is potentially persistent in the environment.


Bioaccumulation:

The Log Pow for NETA is estimated to 3.7 (ref. 8).


Since Log Pow < 4, it indicates that NETA has low potential for bioaccumulation (ref. 1).


Eco-toxicity:

The obtained results of the performed eco-toxicity tests indicate – as stated in section 2 - that norethisterone/NETA:


• is very toxic to the green alga Desmodesmus subspicatus (since EC50 < 1 mg/L)

• has no effect on the immobilisation of Daphnia magna

• has no effect on the growth of the bacteria Pseudomonas putida


Conclusion regarding PBT/vPvB properties:

Considering all three PBT-aspects, NETA does not meet the criteria for classification as a PBT or vPvB substance.


5. Environmental risk classification of NETA


In conclusion:

- A valid risk quotient (PEC/PNEC) for NETA cannot be calculated due to lack of eco-toxicity data.

- NETA:

  • is potentially persistent in the environment

  • has low potential for bioaccumulation

  • is very toxic to green algae

  • does not meet the criteria for classification as a PBT or vPvB substance


Since the PEC/PNEC cannot be calculated due to lack of eco-toxicity data the following environmental risk phrase should be applied to pharmaceutical products containing NETA according to the criteria in ref. 1:


”Risk of environmental impact of norethisterone acetate (NETA) cannot be excluded due to lack of eco-toxicity data”.


6. References


1) Environmental classification of pharmaceuticals at www.fass.se – Guidance for pharmaceutical companies 2012.

2) Research report from Schering, no. X211: Acute immobilization test of norethisterone with Daphnia magna, 02 May 1997.

3) Research report from Schering, no. X224 - draft: Acute immobilization test of norethisterone acetate (ZK 5422) with Daphnia magna, 23 June 1997.

4) Research report from Schering, no. A08345: Growth inhibition test of norethisterone acetate (ZK 5422) on the green algae Desmodesmus subspicatus, 20 January 2004.

5) Research report from Schering, no. X126: Growth inhibition test of norethisterone on the bacterium Pseudomonas putida, 12. aug. 1996

6) Research report from Schering, no. X128: Study on the biodegradability of norethisterone in the CO2-evolution test (modified Sturm-test), 12 Aug. 1996

7) Research report from Schering, no. X308 – Draft: Study on the biodegradability of norethisterone acetate in the CO2-evolution test (modified Sturm test), 17 May 1999.

8) Report from Schering, LJ03.

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