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Long-term toxicity to fish

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Description of key information

OECD 234 – Fish Sexual Development Test (FSDT; see Section 6.1.8. for the Study Summary). The OECD 234 Endpoint Summary is provided in this section because this method is sufficient for fulfilling the long-term toxicity to fish data requirement, so the key value for the chemical safety assessment from this study is utilised for this endpoint. Further, no Endpoint Summary functionality is available in Section 6.1.8 Endocrine Disruptor Testing in Aquatic Invertebrates – in vivo , hence all relevant discussion related to Section 6.1.8 is also included below.

The test was performed on the read-across source substance: 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanol (6:2 FTOH) which is a metabolite/transformation product of 6:2 FTMA

  • A statistically significant effect was observed on hatching success at nominal concentrations of 0.96 mg/L and above.
  • Statistically significant effects were observed on survival and growth (male and female length and weight) at the highest nominal test concentration of 3.0 mg/L.
  • Sex ratio was not affected by the test substance at the concentrations employed with no statistically significant differences observed between the test concentrations and solvent control for either genetic or phenotypic sex ratio.
  • Whilst no statistically significant increase or decrease in VTG concentration in female fish was observed, statistically significant effects were observed for male secondary sex characteristics and male VTG concentration. A significant decrease in the number of anal fin papillae was observed at the 3.0 mg/L test concentration whilst a significant increase in VTG concentration was observed at the 0.30 mg/L test concentration and above.

The overall NOEC covering all endpoints tested was considered to be 0.096 mg/L based on nominal test concentrations (0.0137 mg/L based on geometric mean measured concentrations). A summary of the individual endpoints assessed are given in Table 1 below:

Table 1.  NOEC and LOEC values for all endpoints examined

Response

Nominal concentration
(mg/L)

Geometric Mean Measured Concentration (mg/L)

NOEC

LOEC

NOEC

LOEC

Hatching success

0.30

0.96

0.0231

0.0537

Final survival

0.96

3.0

0.0537

0.0953

Female growth (lengths and weights)

0.96

3.0

0.0537

0.0953

Male growth (lengths and weights)

0.96

3.0

0.0537

0.0953

Genetic sex ratio (% males)

≥3.0

>3.0

≥0.0953

>0.0953

Phenotypic sex ratio (% males)

≥3.0

>3.0

≥0.0953

>0.0953

VTG concentration (female)

≥3.0

>3.0

≥0.0953

>0.0953

VTG concentration (male)

0.096

0.30

0.0137

0.0231

Secondary sex characteristics

0.96

3.0

0.0537

0.0953

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Effect concentration:
0.014 mg/L

Additional information

Endocrine disruptor testing in aquatic vertebrates - in vivo - Endpoint Summary - IUCLID Section 6.1.8 - OECD 234 (Fish Sexual Development Test; FSDT) Summary

A test in accordance with OECD 234 (2011), a FSDT, has been performed in order to assess the endocrine-disrupting potential of the test item (read-across source substance: 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanol), in particular the potential for an estrogenic mode of action was investigated.

The test was initiated by adding fertilised Oryzias latipes eggs to test vessels (120 eggs per concentration; < 12 h old), which were exposed to a range of concentrations of the test substance dissolved in water under semi-static test conditions (with daily renewal of the test media). Due to difficulties encountered with maintaining concentrations with a flow-through system, based on the results of media preparation trials, a solvent spike (acetone) using semi-static (daily) renewal was used as the dosing type for this study. The test was continued until fish were sexually differentiated at 112 days post-hatch (dph) (total duration: 122 days).

The definitive test was conducted at nominal test concentrations of 0.030, 0.096, 0.30, 0.96 and 3.0 mg/L. Control and solvent control groups were also included. Four replicate vessels were used for the controls and each test concentration.

At the end of the exposure, the adult fish were assessed for growth (weight and length), secondary sex characteristics (anal fin papillae count), genetic sex (by PCR method) and vitellogenin (VTG; by ELISA method). In addition, gonad histology was performed to determine the phenotypic sex of the fish. Hatching success and final survival were also examined.

Chemical analysis was conducted weekly throughout the test on freshly prepared media and the corresponding 24 h old media. Despite all measures taken, nominal concentrations were generally not achieved and were not maintained over the 24 h semi-static exposure periods. This was due to the highly volatile nature of the test substance and the requirement to aerate the vessels to maintain fish health. Given the variability in measured concentrations throughout the test, the results have been calculated based on both nominal and mean measured concentrations. The geometric mean measured concentrations were calculated to be 0.00933, 0.0137, 0.0231, 0.0537 and 0.0953 mg/L.

The results of individual endpoints are described in Table 1 in the free text box ‘Description of key information’ above. The overall NOEC covering all endpoints tested was considered to be 0.096 mg/L based on nominal test concentrations, or 0.0137 mg/L based on geometric mean measured concentrations.

Discussion and contextualisation of the FSDT results

Relevance of results to ED guidance/frameworks

  1. OECD Test Guideline (TG) 234 (2011)

Relevance to FSDT results: Relating to identification of potential endocrine activity, the OECD 234 TG states that:

Two core endpoints are measured as indicators of endocrine-associated developmental aberrations: vitellogenin (VTG) concentration and sex ratio determined via gonad histology (para. 2). If both the aforementioned endpoints were positive in the study, it would provide a clear indication of an endocrine MoA (para. 8). However, only one of the two core endocrine endpoints was positive in the 6:2 FTOH study – VTG concentration, in males only. There was no statistically significant effect on sex ratio.

  1. VTG induction in males – possible modality: estrogenic, or aromatisable androgen (para. 4)

Another endocrine-relevant endpoint for which effects were observed was:

  1. Secondary sexual characteristics (SSC) – Minimal information is provided in the OECD 234 TG. However, in the OECD 230 (21-d fish assay), the following is stated: A decrease in SCC in males should be interpreted with caution because of low statistical power, and should be based on expert judgement and weight of evidence (OECD 230, para. 7).
  2. Effects were also observed on hatching success, final survival and growth. These endpoints are not specifically addressed in terms of endocrine-relevance in the OECD 234 TG, but are discussed in Guidance documents as outlined below.

Conclusion: 6:2 FTOH exhibits effects that may plausibly be linked to an endocrine mode of action (MoA), but as only one of the two core endocrine endpoints were positive for effects, it is inconclusive considering the above information in isolation.

2a.     OECD Revised Guidance Document No. 150 (2018) – Conceptual Framework (CF)

Relevance to FSDT results: - notes from OECD No. 150:

  • VTG induction in males: estrogen-mediated activity (agonistic) (Sec B.2, Table B.1).
  • Endpoints potentially sensitive to, but not diagnostic of, E,A,T,S: body length, body weight, (Sec B.2, Table B.1, p60); additionally hatching success is described in Table B.1; but for FLCTT, MEOGRT, ZEOGRT TGs.
  • It should be noted that if the OECD 234 assay gives a positive result, this may be due to a positive indicator of hormonal activity (e.g. VTG), a positive for biased sex ratio, or a positive for both types of endpoint. Each of these three possible combinations of positive response should be considered separately (Sec C.2.9, para. 407).

Conclusion: 6:2 FTOH exhibits effects that may plausibly be linked to an endocrine MoA, particularly when considering that effects on body length and body weight (females and males) and hatching success were statistically significantly affected, as well as VTG concentration in males.

2b.    OECD No 150: Table C.2.9, taking into account the availability of Conceptual Framework (CF) Level 2 and 3 data (see below) as discussed by ECHA in the Decision on Substance Evaluation on 6:2 FTMA (Feb 2019)

Relevance to FSDT results:By placing the available data into context according to the guidance in Table C.2.9 (p281) is as follows:  

Scenario:                                                            A(some pre-existing mechanistic in vitro data available demonstrating positive effects; some pre-existing in vivo data available demonstrating effects of concern; combined with a positive result from an FSDT). This results in the following:  

Result of TG234 (FSDT):                     +  

Existing results:  

Mechanism (in vitromechanistic data):     +  

Effects (in vivoeffects of concern):            +  

Possible conclusion:                                        1) - ‘Indicators of endocrine activity and apical endpoints positive” leading to the outcome ‘Strong evidence for adverse effects in fish and other organisms by an endocrine mechanism.’

Next step which could be taken to strengthen WoE approach if necessary:

Some regulatory authorities may consider that further evidence isnotrequired, especially if adverse effects have been demonstrated. However, if more evidence is needed about adverse effects in fish, performance of a fish life cycle test (MEOGRT: OECD TG 240, or ZEOGRT) should be considered.Other considerations:                                    

Some EDs may be more toxic to reproduction than to sexual development, in which case TG 234 (FSDT) would be less responsive than a life cycle test (MEOGRT or ZEOGRT).

 

Conclusion: 6:2 FTOH exhibits effects that may plausibly be linked to an oestrogenic MoA,when considering the weight-of-evidence available.

1. ECHA/EFSA Guidance for the identification of endocrine disruptors in biocides and pesticides (2018)   Relevance to FSDT results:- notes from ECHA/EFSA Guidance:

  •   VTG induction in males – is an in vivo mechanistic parameter (Table 15, p78); and a decrease in VTG is generally considered EAS-mediated, but can be caused by overt/systemic toxicity and non-ED MoAs (e.g. hepatotoxicity), or diet or infection – therefore the results should be interpreted in combination with other observations (Sec 4.3.2.1, p71).

    SSC - A decrease in SSC in males may indicate an estrogenic or anti-androgenic MoA but can also be influenced by non-endocrine MoA; it should therefore be interpreted with caution and based on WoE and expert judgement (OECD, 2009b – OECD 230) (p72). It is an EAS-mediated parameter in medaka (Table 15, p78).

  • Survival, hatching success, length & body weight – ‘Sensitive to, but not diagnostic of, EATS’ (rather than an EATS-mediated parameter), not assigned to a specific modality (Table 15, p78).
  • Figure 1: Flowchart illustrating ED assessment strategy (p13) –

“Have the E-mediated parameters been sufficiently investigated?” (See point I/II below) --> YES --> “Has E-mediated adversity been observed?” --> YES --> Next step: “postulate MoA(s) considering the adversity and/or endocrine activity identified above”. (See point III. below) [If there is not sufficient information to support the postulated MoA(s), further information must be generated.] “Sufficient info to support the postulated MoA?” --> “Establish biological plausibility between adverse effect(s) and endocrine activity for the postulated MoA”. --> “Does the available evidence support the biological plausibility for an endocrine MoA?” (See point IV. below) --> YES --> STOP: ED criteria ARE MET

I. To consider the EAS-mediated adversity for non-mammalian non-target organisms sufficiently investigated (to conclude on an absence of EAS-mediated adversity), the ‘EAS-mediated’ parameters foreseen to be measured in the MEOGRT should have been investigated and the results included in the dossier. Alternatively, a fish life-cycle toxicity test (FLCTT) covering all the ‘EAS-mediated’ parameters foreseen to be measured in the MEOGRT (see Table 15, p78) is acceptable (Section 3.4.1, p31). Note: All EATS-mediated parameters (see Table 15, p78) required by the MEOGRT TG are measured in the OECD 234.

II. To consider the E, A, T, S modalities for non-target organisms other than mammals sufficiently investigated (to conclude on an absence of EATS-related endocrine-activity), preferably the FSTRA (OECD 229) should have been conducted; however the 21-day fish assay OECD 230 is acceptable as well. If data are already available covering the mechanistic parameters investigated in OECD TG 229 or OECD TG 230 (e.g. OECD TG 234), then those data could be used instead (Sec 3.4.2, p32).

III. MoA postulation:

MoA 1:

Molecular Initiating Event (MIE): Estrogen receptor agonism

Key Event (KE) 1: Increased VTG synthesis in livers of male fish

KE 2: Increased VTG circulating in male fish, slow degradation of VTG (Denslow et al., 1999)

KE 3: Change in male fish gonad histology (decreased size*, decreased spermatogenesis*) (Kidd et al., 2007; Sugawara, 2011)

Adverse outcome: Decreased fecundity or fertility; sexual dysfunction** (AOP No. 29, under development; Cheek et al., 2001)

*Observed in the OECD 234: Fish (females and males) exposed to nominal 0.96 or 3.0 mg/L often had smaller gonads in comparison to controls and lower concentration groups, as well as fewer stages of spermatogenesis/oogenesis present within the tissue. Comparison of genetic sex and phenotypic sex indicated some feminisation of male fish at the highest test concentration of nominal 3.0 mg/L.

**Postulated adverse outcome, these endpoints are not measured in the OECD 234

 

MoA 2:

MIE: Estrogen receptor agonism

KE 1: Increased VTG synthesis in livers of male fish

KE 2: Increased VTG circulating in male fish; slow degradation of VTG (Denslow et al., 1999)

KE 3: Renal toxicity/failure; subsequent disruption to blood dynamics and function^ (Herman & Kincaid, 1988; Sugawara, 2011)

Adverse outcome: Increased mortality^^ (AOP 53, under development)

^ Postulated KE, the kidney is not examined in the OECD 234

^^Observed in the OECD 234: A significant reduction in survival was observed at the nominal 3.0 mg/L test concentration compared to the solvent control.

Relevant supporting information for MoA analysis:

Adverse Outcome Pathway (AOP) No. 29: Estrogen receptor agonism leading to reproductive dysfunction (https://aopwiki.org/aops/29)

AOP No. 53: ER agonism leading to reduced survival due to renal failure (https://aopwiki.org/aops/53)

KE 252 (https://aopwiki.org/events/252)

KE 418 (https://aopwiki.org/events/418)

References:

Cheek et al., 2001. Experimental evaluation of vitellogenin as a predictive biomarker for reproductive disruption. Environmental Health Perspectives, 109(7):681–690

Denslow et al., 1999. Vitellogenin as a Biomarker of Exposure for Estrogen or Estrogen Mimics, Ecotoxicology, 8(5):385-398.

Kidd et al., 2007. Collapse of a fish population after exposure to a synthetic estrogen. PNAS. 104(21): 8897-8901

Herman & Kincaid, 1988. Pathological effects of orally administered estradiol to rainbow trout. Aquaculture. 72:165–172

Sugawara, 2011. Screening systems for endocrine disruptors. Chapter 68, in Reproductive and Developmental Toxicology. 893-902.

IV. In the scenario 2b [as applies for the FSDT], where adversity is based on ‘EATS-mediated’ parameters, the underlying knowledge of the likely endocrine nature of the effects may be such that judgement can be reached on the biological plausibility of a link without recourse to a detailed MoA analysis. In such cases, the MoA analysis could be very simple; when an adverse effect is ‘EATS-mediated’, the biologically plausible link is already pre-established in the absence of information proving the contrary (i.e. a fully developed non-ED MoA). This is because, in the case of ‘EATS-mediated’ parameters, where the pattern of effects is deemed adverse, the biological plausibility that the adverse effects are caused via an EATS-mediated MoA is high, based on existing knowledge and theory, and as such, it may not be necessary to generate further empirical data on the substance under evaluation to substantiate the link between the observed adverse effect(s) and an endocrine-mediated MoA (Sec 3.5.2, p39).

Conclusion

ECHA/EFSA (2018) Section 3(c): Are the adverse effects a consequence of the endocrine MoA? I.e., is there a biologically plausible link between the adverse effect and the endocrine activity, specifically relevant for the population level? (p7). Yes, there is a biologically plausible link.

  1. Pre-existing data on 6:2 FTOH

Relevance to FSDT results: Pre-existing data on the substance have been considered in the context of the FSDT results. A conclusion is provided with the data source, regarding whether the results are in agreement with the FSDT results - i.e., effects observed that are indicative of potential endocrine activity (via an estrogenic modality) caused by the substance or not.

  • Reference: Maras et al. 2006 (in vitro)

Result: Positive

Conclusion: Evidence of potential estrogenic activity - in agreement with FSDT results.

  • Reference: Ishibashi et al. 2007 (in vitro)

Result: Positive

Conclusion: Evidence of potential estrogenic activity - in agreement with FSDT results.

  • Reference: Ishibashi et al. 2008 (in vitro)

Result: Positive

Conclusion: Evidence of potential estrogenic activity - in agreement with FSDT results.

  • Reference: Liu et al. 2007 (in vitro)

Result: Positive

Conclusion: Evidence of potential estrogenic activity - in agreement with FSDT results.

  • Reference: Ishibashi et al. 2008 (in vivo)

Result: Positive

Conclusion: Evidence of potential estrogenic activity - in agreement with FSDT results.

  • Reference: Liu et al. 2009 (in vivo)

Result: Positive

Conclusion: Evidence of potential estrogenic activity - in agreement with FSDT results.

  • Reference: Benninghoff et al. 2011 (in vitro)

Result: Negative

Conclusion: Evidence of potential estrogenic activity – not in agreement with FSDT results.

  • Reference: Benninghoff et al. 2011 (in vivo)

Result: Positive

Conclusion: Evidence of potential estrogenic activity - in agreement with FSDT results.

  • Reference: Goodband, 2021 (in vivo - FSDT)

Result: Positive

Conclusion: Evidence of potential estrogenic activity – the FSDT mostly in agreement with the experimental conclusions described in the references above. Effects were observed on male SSC (decreased number of anal fin papillae), male VTG (but not female VTG), female and male body length and body weight, hatching success and survival. Increased VTG in males is in vivo mechanistic evidence of the estrogen modality. Decreased number of anal fin papillae (SSC) is an EATS-mediated parameter, evidence of estrogen or possibly anti-androgen modality. The estrogen modality is considered more likely due to male VTG effects in conjunction with the weight of evidence presented above, i.e. pre-existing Conceptual Framework (OECD, 2018) L2 & 3 data.

  1. Conclusion

The FSDT was requested by ECHA in a Decision on Substance Evaluation (2019),  to in order to conclude on population relevant adverse effects and to conclude on the endocrine disrupting properties on 6:2 FTOH.

In terms of population relevance:

Impacts on growth, as observed in the FSDT, are generally regarded as population relevant (European Commission, 2011; in EFSA/ECHA, 2018, Sec 3.3.1.4).

Sex ratio, which is both a biomarker and an apical endpoint in the OECD 234 and is considered population relevant (OECD No. 150, 2018, Sec C.2.9, para. 407), did not exhibit statistically significant effects in the FSDT.

Effects on survival, as observed in the FSDT, is considered to be a population relevant adverse effect (OECD TG 240, 2015, para. 2). Effects on hatching success, as observed in the FSDT, is considered to be a population relevant adverse effect (OECD TG 240, 2015, para. 33).

For SSC, for which effects were observed in the FSDT, there is ongoing debate on the consideration of SSC as an apical endpoint and about the relevance of this endpoint at thepopulation level (ECHA/EFSA, 2018, Sec 4.3.2.1, p72).

In conclusion, the weight-of-evidence available indicate that the substance has estrogenic properties, although only one (VTG) of the two core endocrine endpoints in the OECD 234 (VTG and sex ratio) demonstrated significant effects in the FSDT performed on the substance. However, the significant reduction observed in male SSC at the top two tested concentrations supports the postulated estrogenic MoA. Further, statistically significant effects were observed on survival, male and female growth and hatching success. These parameters are classified as ‘sensitive to, but not diagnostic of, EATS’, thus contributing support to the notion of endocrine activity (but these parameters are not indicative of a specific modality). Further, some feminisation of male fish at the highest tested concentration was observed; as well as smaller gonads in fish when compared to controls and lower concentration groups, and fewer stages of spermatogenesis/oogenesis present in tissue at the top two concentrations.

The evidence indicates that the substance displays agonistic effects on the estrogen receptor, from the increased male VTG concentrations and reduced male SSC, observed effects on gonad histology and supported by estrogenic mechanistic data from L2 and L3 studies.

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