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Ecotoxicological information

Endpoint summary

Administrative data

Description of key information

OECD 231 (See Section 6.1.8. for study summary. No endpoint summary functionality is available in Section 6.1.8 for this endpoint, therefore the summary is provided here): At 107 mg test item/L (the top dose; day 21): a statistically significant increase compared to the control was observed in normalised hind limb length and developmental stage (advanced development observed).

At 1.07 mg test item/L, no statistically significant difference compared to the control was observed for any endpoint. At 10.7 mg test item/L, wet weight and snout vent length were statistically significantly increased compared to the control on day 7, whereas on day 21 no statistically significant difference compared to the control was observed for any endpoint. At 107 mg test item/L on day 7, no statistically significant difference regarding developmental stage was detected; however, statistically significant advanced development on day 21 was observed. Body wet weight and snout vent length were statistically significantly increased on day 7 of exposure, but not on day 21. Under the conditions of this study, there were no changes in the thyroid glands that could be attributed to treatment with the test item.

Additional information

Endocrine disruptor testing in aquatic vertebrates - in vivo - Endpoint Summary - IUCLID Section 6.1.8 - OECD 231 (Amphibian Metamorphosis Assay)

Summary

A test in accordance with OECD 231 (2009) was performed on PFHxA in order to assess the thyroid activity potential of the test item. See Section 6.1.8 for the OECD 231 robust study summary.

Fertilised Xenopus laevis (African clawed frog) eggs were hatched and the tadpoles reared until they reached NF (Nieuwkoop & Faber, 1994) developmental stage 51. The organisms were then exposed in a flow-through test for 21 days. There were 4 treatment groups (3 concentrations of test item and 1 negative control), with 4 replicates each containing 20 organisms. The test concentrations used were 1.07, 10.7 and 107 mg test item/L and a control, corresponding to 1.0, 10 and 100 mg perfluorohexanoate/L. Analytical verification of the test item concentration in test medium was performed (the overall mean recovery value was 99 %).

The endpoints assessed in the study were developmental stage (day 7 and 21); body weight (day 7 and 21); snout to vent length (SVL; day 7 and 21); hind limb length (HLL; day 7 and 21) thyroid histology (day 21) as indicators of thyroidal activity of the test item. Furthermore, survival and behaviour were monitored daily.

Key results: At 107 mg test item/L, statistically significant advanced development on day 21 was observed compared to the control. A statistically significant increase of HLL (normalised by SVL) at 107 mg test item/L on day 7 and 21 were observed compared to the control.

At 1.07 mg test item/L, no statistically significant difference compared to the control was observed for any endpoint. At 10.7 mg test item/L, wet weight and snout vent length were statistically significantly increased compared to the control on day 7, whereas on day 21 no statistically significant difference compared to the control was observed for any endpoint. At 107 mg test item/L on day 7, no statistically significant difference regarding developmental stage was detected; however, statistically significant advanced development on day 21 was observed. Body wet weight and snout vent length were statistically significantly increased on day 7 of exposure, but not on day 21. Under the conditions of this study, there were no changes in the thyroid glands that could be attributed to treatment with the test item.

 

Discussion and contextualisation of the AMA results

Relevance of results to endocrine disruption guidance/frameworks

  1. OECD Test Guideline 231 (2009)

Relevance to AMA results: Relating to potential thyroid activity, the OECD 231 Test Guideline (TG) states that:

  • Advanced development (as observed in the AMA study) is only known to occur through effects which are thyroid hormone-related. This is considered sufficient evidence to indicate that the chemical has thyroid activity.
  • If statistically significant advances in development or hind limb length occur, then the test indicates that the chemical is thyroid active.
  • Hind limb length, at both days 7 and 21, is positively associated with agonistic effects on the thyroid hormone receptor.

Conclusion: PFHxA would be considered thyroid active (specifically, a thyroid hormone receptor agonist) based on the OECD 231 TG criteria. No conclusion on endocrine disruption can be made.

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

Relevance to AMA results: Relating to potential thyroid disruption, OECD No. 150 states that:

  • If only an apical endpoint responds in the AMA - (i.e. no positive histopathological endpoints; this scenario reflects the results from the AMA performed on the substance), it suggests that the chemical is a possible thyroid disrupter. However, there is somewhat reduced confidence in this conclusion in some cases, compared to a scenario in which only histopathological effects were observed (although existing positive in vitro data would be weighed against this conclusion*). However, apical endpoints alone are probably sufficiently responsive to thyroid receptor agonists (p213, paragraph 319).
  • Although a positive response from an OECD 231 study indicates that the chemical is a possible thyroid disrupter, a result of this type would generally need to be followed up with a more comprehensive growth, development and/or reproduction test if further evidence was needed.

*In vitro data on PFHxA are available and summarised later in this Discussion. Four of the five studies discussed in the ECHA Decision on Substance Evaluation Letter (SEV-241-527-8-1_DEC_FINAL) indicate potential concern for thyroid activity; the fifth was negative for potential thyroid activity.

Conclusion: PFHxA is a possible thyroid disrupter, a more comprehensive test would be required in order to make a more robust conclusion. No conclusion on endocrine disruption can be made.

        2b. OECD Revised Guidance Document No. 150 (2018) – CF: Table C.2.3:

Relevance to AMA results: Relating to potential endocrine activity based on existing data, the OECD No. 150 Table C.2.3. applicable scenario is:

  • C - 'some pre-existing mechanistic in vitro data available; no pre-existing in vivo data available; combined with a positive AMA result'.
    • Point (3): ‘Some evidence for in vivo thyroid activity with potential adverse effects (developmental/growth toxicity) in amphibians.

Conclusion: There is some evidence that PFHxA may be thyroid active, a more comprehensive test would be required in order to make a more robust conclusion. No conclusion on endocrine disruption can be made.

        3. EFSA/ECHA Guidance for the identification of endocrine disruptors in biocides and pesticides (2018)

Relevance to AMA results: Relating to potential endocrine disruption; from the ECHA/EFSA document, following Figure 1: Flowchart illustrating ED assessment strategy (p13):

“Has the thyroid-mediated parameter been sufficiently investigated?” -> No: “Has thyroid-mediated adversity been observed?” -> Yes -> “postulate MoA(s) considering the adversity and/or endocrine activity identified above”. If there is not sufficient information to support the postulated MoA(s), further information must be generated.

Conclusion: No conclusion on endocrine disruption can be made, without generating further information.

Pre-existing data on PFHxA

Pre-existing data on the substance have been considered in the context of the AMA results. A conclusion is provided with data source, regarding whether the results are in agreement with the AMA results - i.e., potential thyroid activity caused by PFHxA or not.

 

  1. Reference: Loveless et al., 2009 (in vivo)

Result: Positive

Conclusion: Evidence of potential thyroid activity - in agreement with AMA results.

  1. Reference: Weiss et al., 2009 (in vitro)

Result: Positive

Conclusion: Evidence of potential thyroid activity - in agreement with AMA results.

  1. Reference: Vongphachan et al., 2011 (in vitro)

Result: Positive

Conclusion: Evidence of potential thyroid activity - in agreement with AMA results.

  1. Reference: Naile et al., 2012 (in vitro)

Result: Positive

Conclusion: Evidence of potential thyroid activity - in agreement with AMA results.

  1. Reference: Kim et al., 2015 (in vivo)

Result: Positive

Conclusion: Evidence of potential thyroid activity - in agreement with AMA results.

  1. Reference: Ren et al., 2016 (in vitro)

Result: Positive

Conclusion: Evidence of potential thyroid activity - in agreement with AMA results.

  1. Reference: Li et al., 2017 (in vivo)

Result: Positive

Conclusion: Evidence of potential thyroid activity - in agreement with AMA results.

 

  1. Reference: Kirkpatrick, 2005 (in vivo)

Result: Negative

Conclusion: No evidence of potential thyroid activity - not in agreement with AMA results.

  1. Reference: Chengelis et al., 2009 (in vivo)

Result: Negative

Conclusion: No evidence of potential thyroid activity - not in agreement with AMA results.

  1. Reference: Frey et al., 2010 (in vivo)

Result: Negative

Conclusion: No evidence of potential thyroid activity - not in agreement with AMA results.

  1. Reference: Cassone et al., 2012 (in vivo)

Result: Negative

Conclusion: No evidence of potential thyroid activity - not in agreement with AMA results.

  1. Reference: Klaunig et al., 2015 (in vivo)

Result: Negative

Conclusion: No evidence of potential thyroid activity - not in agreement with AMA results.

  1. Reference: Ren et al., 2015 (in vitro)

Result: Negative

Conclusion: No evidence of potential thyroid activity - not in agreement with AMA results.

  1. Reference: ToxCast/Tox21 Database (summarised in Borghoff et al., 2018)

Result: Negative (4/4 assays)

Conclusion: No evidence of potential thyroid activity - not in agreement with AMA results.

 

To summarise, there are available data to suggest that PFHxA may potentially cause thyroid disruption and data to suggest that PFHxA does not cause thyroid disruption.

 

Conclusion

The weight-of-evidence as discussed above indicates that PFHxA has thyroid active properties, however it is difficult to conclude on the specific mode of action without further information. Evidence from the AMA performed on the substance indicates no direct effects on the thyroid gland itself. Rather, the substance exhibits agonistic effects on thyroid hormone receptors, from enhanced development in the AMA and supported by thyroid-related mechanistic data from in vitro studies outlined above.  Although the responses seen in the AMA were relatively subtle, the results meet the criteria for thyroid activity in the OECD 231 TG. No assessment of potency can be made at this stage - a conclusion on potency would require a CF Level 4 or 5 study as prescribed in OECD No. 150 (2018).

In conclusion, based on the results obtained from the AMA performed on PFHxA in conjunction with the pre-existing data available for PFHxA, whilst it is possible to conclude that the substance has thyroid activity potential, it is not possible to conclude on whether the substance is an endocrine disruptor.