Phenol, mono- + distyrenated (MSP+DSP)

Additional ecotoxological information

Administrative data

Endpoint:

additional ecotoxicological information

Remarks:

Fish Sexual Development Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-02-2018 to 26-04-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 234 Fish Sexual Development Test

Version / remarks:

July 2011

Deviations:

yes

Remarks:

Minor deviations in test temperature and mean measured concentrations. These deviations did not influence the overall validity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study / information:

Early life stage toxicity and sexual development in fish

Test material

Test material information

Constituent 1

Reference substance name:

p-(1-phenylethyl)phenol

EC Number:

217-864-1

EC Name:

p-(1-phenylethyl)phenol

Cas Number:

1988-89-2

Molecular formula:

C14H14O

IUPAC Name:

4-(1-phenylethyl)phenol

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RÜTGERS Germany GmbH, Varziner Strasse 49, 47138 Duisburg (Germany)
- Lot/Batch n°: Lab Sample 6.9.2017
- Expiration date of the lot/batch: 31-12-2018
- Purity test date: 06-09-2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions of test material: room temperature, dry
- Storage stability of the test item in frozen samples: Spiked samples frozen on 15-01-2018 were thawed 26-04-2018 and analysed alongside the t63 samples.
The analysis of the thawed samples confirmed the storage stability.SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RÜTGERS Germany GmbH, Varziner Strase 49, 47138 Duisburg (Germany)
- Lot/Batch n°: Lab Sample 6.9.2017
- Expiration date of the lot/batch: 31-12-2018
- Purity test date: 06-09-2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions of test material: room temperature, dry
- Storage stability of the test item in frozen samples: Spiked samples frozen on 15-01-2018 were thawed 26-04-2018 and analyzed alongside the t63 samples.
The analysis of the thawed samples confirmed the storage stability.

Results and discussion

Any other information on results incl. tables

A. Sex ratio

Table 1 Biological effects at test termination (63 dpf) (excerpt: endocrine effects only, from amended Report 2019, Table 11, p. 12)

(see also Diagram in Attached Document)

		Nominal concentration 4 -(1 -phenylethyl)-phenol [μg/L]
		Control	2.0	6.3	20.0	63.0	200.0
		Mean measured concentration 4-(1-phenylethyl)phenol [μg/L]
		Control	2.1	6.4	19.7	61.8	187.9
No of replicates		4	4	4	4	4	4
Sex ratio [% females]*	Mean	60.2	57.9	49.5	41.2	50.1	36.2
	SD	5.8	18.0	7.3	10.3	10.2	12.5
	RSD	9.6	23.1	14.8	24.9	19.9	34.4
Sex ratio [% males]*	Mean	28.9	28.2	30.0	24.4	18.9	0.0***
	SD	15.1	6.8	3.2	15.7	6.5	0.0
	RSD	52.4	24.0	10.5	70.2	34.5	-
Sex ratio [% undifferentiated]*	Mean	10.8	13.9	20.5	36.4**	30.1**	63.8**
	SD	10.7	13.1	4.8	11.2	14.3	12.5
	RSD	98.9	94.4	23.6	30.7	47.5	19.5

dpf = days post-fertilization; SD = Standard deviation; RSD = Relative standard deviation

* Only mature fish with clearly identified sex (histopathology) were considered for evaluation. Fish, which could not be determined histopathologically (unidentified)
or were undifferentiated, were excluded from calculation. Fish defined as ‘unidentified - no gonads’ were not considered for determination of the sex ratio.

** Williams t-test, p<0.05; one-sided greater

*** Jonckheere-Terpstra test, p<0.05; one-sided smaller

Sex ratio:

The sex ratio was determined based on histopathological examinations of the gonads. The examined fish were assigned to three different categories: female, male, or undifferentiated. The category ‘undifferentiated’ described individuals, where the gonads showed only undeveloped ovaries with oogonia to perinucleolar oocytes [7]. The gonads of these animals were most probably arrested in the develop

ment into testis due to the treatment with the test item, and afterwards remained at an early ovary stage. Furthermore, it described fish in transition phase, with gonads consisting of undeveloped germ cells to oogonia exclusively. For these animals, it was not possible to confirm the sex. Individuals, which were assigned as ‘unidentified – no gonads’, were taken out of the calculation, as these individuals could not be sexed due to technical errors during cutting and slide preparation. Individuals compiled in this group did not represent a true separate sex category.

For determination of statistically significant differences, the percentage of mature males and females was considered. Furthermore, statistical differences with respect to the percentage of undifferentiated fish were calculated.

In controls and in the four lower test concentrations, the mean value of %males ranged between 18.9% at 61.6 μg 4-(1-phenylethyl)-phenol/L (mean measured concentration) and 30.0% at 6.4 μg 4-(1-phenylethyl)-phenol/L(mean measured concentration). No mature males were found in any of the replicates at the mean highest test concentration of 188 μg/L. Thus, a statistically significant difference (p<0.05) in the sex ratio in terms of %males was observed for the highest test concentration. The percentage of females ranged between 36.2% at 188 μg 4-(1-phenylethyl)-phenol/L (mean measured concentration) and 60.2% (control). No statistically significant differences was thus observed.

There was a concentration-related, statistically significant increase (p<0.05) in the mean value of undifferentiated fish from the concentration of 6.4 μg 4-(1-phenylethyl)-phenol/L. In controls, the mean value for undifferentiated fish was at 10.8%, and increased concentration-depended to 63.8 % at the highest mean test level at 188 μg 4-(1-phenylethyl)-phenol/L.

The NOEC for the endpoint sex ratio (%males) was defined as 61.8 μg/L. Regarding the number of undifferentated fish, the NOEC was defined as 2.1 μg 4-(1-phenylethyl)-phenol/L. Single immature oocytes were found in the testis of some individuals (testis-ova; compare Annex A.6), which was defined as pathological alteration of the testis tissue. These individuals are however undoubtedly identified as

males and not assigned to the category of ‘intersex’.

--------------------------------------------------------------------------------------

B. Vitellogenin (VTG) content in blood plasma

VTG content in females and males(63 dpf) (from amended Report 2019, Table 12, p. 13)

		Nominal concentration 4-(1-phenylethyl)-phenol [μg/L]
		Control	2.0	6.3	20.0	63.0	200.
		Mean measured concentration 4-(1-phenylethyl)phenol [μg/L]
		Control	2.1	6.4	19.7	61.8	187.9
No of replicates		4	4	4	4	4	4
VTG / total protein [ng/μg]; females*	Mean	480.8	407.8	500.3	404.5	451.9	729.7**
	SD	204.0	82.2	65.5	163.4	181.2	307.4
	RSD	42.4	20.2	13.1	40.4	40.1	42.1
VTG / total protein [ng/μg]; males*^#	Mean	0.07	0.39	0.77	0.04	0.07	-
	SD	0.02	0.61	1.04	0.02	0.08	-
	RSD	27.9	158.2	134.6	52.9	108.4	-

VTG / total protein [ng/μg]; undifferentiated*	Mean	0.7	92.01	2.37	2.40	21.30	319.69***
	SD	0.91	183.11	2.43	2.83	25.88	129.31
	RSD	130.3	199.0	102.7	118.0	121.5	40.5

dpf = days post-fertilization; SD = Standard deviation; RSD = Relative standard deviation

* Only mature fish with clearly identified sex (histopathology) were considered for evaluation.

** Williams t-test, p<0.05; one-sided greater

*** Jonckheere-Terpstra test, p<0.05; one-sided smaller

# No values could be obtained for the highest concentration as no mature male fish could be identified.

The VTG content in the blood plasma of all individuals was determined. However, only the values of mature males and females were considered for analysis. Fish, which could not be determined histopathologically (unidentified – no gonads), were excluded from calculation.

A statistically significant increase in mean VTG values was reported in females, only at the highest test concentration (188 μg/L). There were no significant differences in VTG contents for males at any concentration level.

Of note, VTG content could not be determined at the highest concentration as no mature males were present. A statistically significant increase in mean VTG values at the highest treatment level (188 μg 4-(1-phenylethyl)-phenol/L) was also determined for undifferentiated fish. Based on the results for mature females and for undifferentiated fish, the NOEC for the biomarker VTG was determined at 61.8 μg/L (mean measured concentration).

Applicant's summary and conclusion

Conclusions:

Interpretation of the obtained results (an increase in the vitellogenin (VTG) content in females as well as the absence of males in the highest test concentration) suggests a strong estrogenic mode of action for the test substance. This interpretation follows the OECD TG 234. Based on these endocrine-related endpoints, the following NOEC was determined (based on the measure %males, corresponding to an increase in the measured VTG concentration in female blood plasma): NOEC of 61.8 μg/L 4-(1-phenylethyl)phenol (mean measured concentration).

Executive summary:

A fish sexual development test (FSDT) was performed with zebrafish (Danio rerio). The objective of this study was the assessment of effects of continuous exposure to 4-(1-phenylethyl)phenol (4-MSP) on the early life stages and sexual differentiation of zebrafish (Danio rerio), following the OECD test guideline 234. (see also IUCLID-Section 6.1.2: Early Life Stages).

The study was conducted with nominal concentrations of 2.0, 6.3, 20.0, 63.2 and 200 μg/L in four replicates each under flow-through conditions. An untreated control was run in parallel. Exposure was started with 30 fertilised eggs per test vessel and replicate. Biological effects were based on mean measured concentrations (2.1 μg/L; 6.4 μg/L; 19.7 μg/L; 61.8 μg/L;
 187.9 μg/L).

Endpoints which were not indicative of endocrine-mediated effects included hatching success and rates, and mortalities during the early life stage and the juvenile growth. At day 35 post-fertilisation (pf) and when groups were terminated (day 63 pf), fish were digitally photographed. Fish lengths were determined by evaluating photographs using electronically supported analysis. Single wet weights (blotted dry) were determined on day 63 pf (test end).

Endpoints which are indicative of endocrine-mediated effects and which could be determined during the study were the sex ratio and the vitellogenin (VTG) concentration in blood plasma. Sex ratios were determined macroscopically by inspection of the gonads and by histopathological verification in the fish gonads.

Endocrine related endpoints and biomarkers: Sex ratio (based on histopathology)

When compared to controls, a statistically significant test-item-related effect on sex ratio in terms of %males was reported at 187.9 μg/L. No mature males were found in any of the replicates of the highest test concentration, whereas the mean value of %males ranged between 18.9% and 30.0% in controls and the four lower test concentrations. Hence, the NOEC for the endpoint sex ratio (%males) was defined as 61.8μg/L (mean measured concentration).

The mean value of undifferentiated fish ranged between 10.8% (controls) and 63.8% at 188 μg 4-(1-phenylethyl)phenol/L, with a monotonous concentration-response relationship. A statistically significant difference was observed for concentrations ≥6.4 μg 4-(1-phenylethyl)phenol/L.

Regarding the number of undifferentiated fish, the NOEC was defined as 2.1 μg 4-(1 -phenylethyl)phenol/L (mean measured concentration).

Endocrine-related endpoints and biomarkers: Vitellogenin (VTG) content in blood plasma

A statistically significant test item-related increase in vitellogenin level was reported at 188 μg/L in females only (mean VTG value of 729.7 ng/μg protein vs. 480.8 ng/μg protein in controls).

VTG contents for males could be determined only for controls and for the four lower test concentrations, as no mature males were present at the highest test level. No significant differences in VTG content were observed.

A statistically significant increase in mean VTG values at the highest treatment level (188 μg 4-(1-phenylethyl)-phenol/L) was also determined for undifferentiated fish, from 0.70 ng VTG/μg protein in controls to 319.69 ng VTG/μg protein at the highest treatment level.

Based on the results for mature females, the NOEC for the biomarker VTG was determined at 61.8 μg /L (mean measured concentration).

Conclusion

Based on these endocrine-related endpoints, the following NOEC was determined (based on the measure %males, corresponding to an increase of the measured VTG concentration in female blood plasma):  NOEC of 61.8 μg/L 4-MSP (mean measured concentration), corresponding to NOEC of 63.0 μg/L 4-MSP (nominal concentration).