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additional ecotoxicological information
vitellogenin stimulation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-04-14 to 2016-07-04 with the definitive exposure phase from 2016-04-14 to 2016-05-26
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
detailed study adopted from OECD TG 229 with modifications
Reason / purpose for cross-reference:
reference to same study

The bioaccumulation potential of the substance Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol (OAPP, previously phenol, methylstyrenated, Novares LA 300) has been assessed in bioaccumulation/ bioconcentration studies in fish. The BCF of two individual constituents of OAPP, one methylstyrenated (cumyl) substituted phenol and a dimer of 2-phenylpropene were experimentally shown to be low to moderate. For dimers and trimers of 2-phenylpropene, BMF were determined. Conversion into BCF indicates that the BCF for dimers is between 2000 and 5000, while the BCF for trimers is above 5000. However, the conversion of BMF into BCF values show such a high variability/standard deviation due to uncertainties in conversion methods that the high BCF values resulting from conversion have to be considered as not very reliable. They are not suited for assessment and should be taken with caution.

BCF (aquatic species):
3 000 L/kg ww
BMF in fish (dimensionless):

Aquatic bioaccumulation of OAPP and its constituents has been investigated in three bioconcentration/bioaccumulation studies. The constituents diphenylmethylpentene (4-methyl-2,4-diphenylpent-1-ene) (NITE 2002) and cumylphenol (1-methyl-1-phenylethyl)phenol (NITE 1990) were subject of valid bioconcentration flow-through fish tests (OECD 305). BCFs were determined to be 2767/2320 L/kg fish ww (low/high exposure concentration, lipid normalised) and 168 L/kg fish ww (high exposure concentration) at steady state, respectively. In a bioaccumulation study (dietary exposure, OECD 305-III) (Klix 2018), BMF factors could only be determined for the non-phenolic constituents of OAPP. For the phenolic components, the concentrations in fish during the depuration phase was too low to be determined. A bioaccumulation potential was thus found only for the non-phenolic constituents of OAPP. Lipid-based and growth-corrected BMF values were 0.0737 and 0.1374 for dimers and trimers, respectively. Conversion into BCF values by calculation of k1rate constants using various methods resulted in average values of 2564 ± 1803 and 22338 ± 23353 (mean ± SD) for dimers and trimers, respectively. Their variation is very high and the results differ very much, so that especially the higher value is considered not to be reliable. This estimation of k1 bears a high uncertainty indicated by the high standard deviation.

Overall, BCF are below 3000 for about 75 % (w/w) of the test substance (phenolic constituents and dimers) while for approx. 25 % (trimers) a reliable BCF value suited for assessment is not yet available. An estimate derived from the experimental BMF suggests that a BCF for trimers may be above 5000. Based on currently available evidence a preliminary BCF for OAPP is set at 3000 L/kg ww.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
other: OECD TG 229
Version / remarks:
Vitellogenin Induction Assay for detection of Endocrine Disrupting substances, adopted from OECD Guideline 229 (2009)
. See details in the section on ‘Principles of method if other than guideline’.
Principles of method if other than guideline:
The study was conducted as a combined study intended to encompass the two endpoints of bioaccumulation and endocrine (estrogenic) activity (ECHA decision 2014). Exposure was for 14 days via food spiked in parallel with the test material and positive controls, HCB (for bioaccumulation) and 17-beta-estradiol (for estrogenic activity).
The test organisms used were older (12-13 months) than the recommended 20 weeks (approximately 5 months), the exposure duration was 14 days instead of 21 days, and in combination with a dietary route of exposure. A single exposure level had been employed instead of a dose – response exposure regime. In addition, no spawning equipment had been introduced, and no further endocrine activity related parameter beyond Vtg stimulation had been measured nor quantified.
The vitellogenin (Vtg) concentration in blood samples of fish was measured using a vitellogenin-ELISA-assay, where the concentration of Vtg was directly related to the absorbance value at 437 nm. The test was adopted from OECD TG 229 using a commercially available assay kit (ELISA kit, product code V01018401, Biosense Laboratories A/S Bergen, Norway). Briefly, 3 dilution levels of the blood were tested in duplicate (in the range of 1:50 – 1:5000 – 1:50000) with a buffer.
The assay was conducted as described in the assay description (see Appendix), with two deviations:
- For washing of the wells, 200 µL of buffer were used.
- Absorbance measurement was conducted at 437 nm wavelength.
According to the assay description (Vtg SOP, product code V01018401, Biosense Laboratories A/S Bergen, Norway), the Vitellogenin concentration should be calculated by regression analysis of the Vtg standards of different nominal concentrations used on each 96-well plate. For this purpose, 11 standards of different Vtg concentration were tested and used for evaluation. Due to the high background absorbance (NSB) of the used 96-well plates, only the highest 2 – 3 Vtg standards resulted in values > 0 after background correction. Therefore, a reliable and significant regression analysis was not possible. Instead, the mean absorbance of the two tested replicates of every blood sample was used for direct evaluation without further estimation of the Vtg concentration. If not stated otherwise, the dilution level of 1:50 was used for evaluation, and the absorbance background of the respective 96-well plate was subtracted from the mean absorbance of the sample.
GLP compliance:
Type of study / information:
Vitellogenin Induction Assay adopted from OECD TG 229.

Test material

Chemical structure
Reference substance name:
Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol
EC Number:
Cas Number:
Molecular formula:
not applicable
Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol
Test material form:
liquid: viscous
Details on test material:
- Manufacturers identification: Novares LA 300
- Substance type: organic
- Test material is 'Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol' (OAPP), EC list number 700-960-7 (assigned manually to validated substances from inquiries by ECHA). Originally the substance phenol, methylstyrenated, CAS No. 68512-30-1, EC No. 270-966-8 was submitted for registration. Subsequent to substance validation, the identity of the substance was changed by ECHA.
- for additional information see respective study record
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares LA 300, Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol
- Appearance: light yellow to colourless liquid
- Source and lot/batch No.of test material: RÜTGERS Novares GmbH, batch No. 38900
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LA 300_phenol, methylstyrenated
- Expiry date: 2017-02-13

Results and discussion

Any other information on results incl. tables

Vitellogenin concentration as a marker for endocrine disruption

To determine the potential endocrine disrupting properties of the test material, fish had been exposed via food to 500 µg test material/g food for 14 days. The concentration of vitellogenin was measured in fish blood at the beginning and after 7 and 14 days of exposure. Since there was high background interference in the ELISA assay, the vitellogenin concentration cannot be expressed quantitatively, but are given semi-quantitatively as absorbance values only (Table 1). There was evidence of vitellogenin increase in male fish in the test material treatments compared to the controls, while no effect was seen in female fish. In the reference treatment, the male and female fish showed significant increases in Vtg induction compared to control after 14 days of exposure.


Table 1. Summary of Vitellogenin Induction after 14 days in the controls, reference material and test material exposures.


Control Group

Reference Group

Test Material Group







Mean Absorbance*







Absorbance Range

0 – 0.0760

0 – 1.83

1.68 – 2.55

2.45 – 3.11

0 – 3.07

0 – 2.16


[µg/g Food]



100 1

100 1

500 2

>500 2


[µg/g Food]

<100 1

<100 1

<500 2

500 2

n.a.       = not applicable

1             = 17β-estradiol

2             = test material

*            = Statistical difference to control, evaluated by ANOVA.


Mortality of test organisms/ - Mortality and/or behavioural abnormalities of control:

No significant effects on mortality, behaviour, feeding or morphology were observed in the test material or control treatments. In the reference material treatments, a cumulative mortality of 18.2 % was observed between days 10 and 42 of the whole experiment with additional morphological effects (internal bleeding) between day 11 and 13 of the uptake phase. The combination of the two reference materials (HCB and 17β-estradiol) has not been tested before, so interferences (positive or negative) between the two substances cannot be ruled out. 


Observations on body length and weight:

The growth and weight of fish did not change significantly throughout the experiment (both uptake and depuration phase), and it did not differ between treatments.


Analytical details

The test material constituent concentrations were confirmed in the feed and media. In the feed the concentrations were between 90-100 % nominal.

Applicant's summary and conclusion

The study found a significant difference between Vtg induction capacity in male fish exposed to the test material via food when compared to the control. The elevated vitellogenin levels in the reference group indicate the acceptability of the combined test design in regard of endocrine disrupting properties, though on a qualitative basis only. However, due high background interference in the assay, the results were considered only semi-quantitative (i.e. only absorbance values were presented). As the administration of the test material was via a single concentration in food only, no conclusions can be drawn on potential potency of the test material.
Executive summary:

Vitellogenin (Vtg) induction was measured in fish exposed to test material for 14 days via a dietary exposure, in combination with a dietary bioaccumulation study in fish (OECD 305) [see also entry in IUCLID 5.3.1. The concentration of Vtg was measured using an ELISA kit based on OECD TG 229. The results indicated the test material induced Vtg production in male specimen, however, due to background interferences in the assay, the results were regarded semi-quantitative only.

Due to the nature of the test design, i.e. a study combined with the determination of a bioaccumulation potential (OECD 305, dietary version), only a single concentration was tested, so firm quantitative effects data suitable for risk asseement cannot be derived from this procedure.