4,4'-Isopropylidenediphenol, ethoxylated, esters with fatty acids, coco

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For precautionary reasons the substance is classified as Muta. 2, even as the micronucleus study is ambiguous.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-10 to 2018-07-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016-07-29

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
R-127 / batch 180131
- Expiration date of the lot/batch:
2019-01-31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Dry and dark at ambient temperature (10 – 30 °C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
no

Species / strain / cell type:

other: Chinese hamster, ovary fibroblast cell line

Details on mammalian cell type (if applicable):

CELLS USED
The test system used in the study was CHO-K1, Cricetulus griseus (Chinese hamster) ovary fibroblast cell line (ATCC CCL-61), passage 6.
The karyotype features of the test system CHO-K1 is 2n = 22.

MEDIA USED
CHO-K1 cells was maintained in the complete growth medium (Ham’s F12K medium-Kaighn’s modification with 10% fetal bovine serum and 100 u/ml Penicillin and 100 µg/ml of Straptomycin) in exponentially proliferating status at 37oC, 5% CO2.

Metabolic activation:

with and without

Metabolic activation system:

S) mix

Test concentrations with justification for top dose:

5 µl/ml. Experience with negative and positive control substances.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: Good solubiliy in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine

Details on test system and experimental conditions:

Cell exposure procedure

After incubating the subculture of CHO-K1 cells for about 24 hrs, the complete growth medium was removed and immediately replaced by treatment medium (Ham’s F12K medium-Kaighn’s modification with 100 u/ml Penicillin and 100 µg/ml of Streptomycin). The test, negative control and positive control substances were added the treatment medium, respectively.

Exposure was performed with and without metabolic activation for 3 hours, and without metabolic activation for 20 hours (1.5 to 2.0 normal cell cycles). A co-factor supplemented post-mitochondrial fraction (S9) from the liver of Aroclor 1254 induced Sprague-Dawley rats was used as metabolic activation system. The concentration of S9 fraction in S9 mixture was 10%.

After 3- hr cell exposure, the treatment medium was removed; the cells were washed, added with fresh complete growth medium and cytoB at the concentration of 3 µg / ml for another 18- 20 hrs (i.e.,1.5- 2.0 normal cell cycles after the beginning of treatment).

For 20- hr cell exposure (1.5- 2.0 normal cell cycle), the cells were treated in the treatment medium and cytoB at the concentration of 3 µg / ml for 20 hours (1.5- 2.0 normal cell cycles).

Rationale for test conditions:

Guideline OECD 487

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity at high dose level at 20 h exposure without S9 mix is 60 %

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the above study, using 5.0 µl/ml, 1.7 µl/ml and 0.6 µl/ml of test item (dissolved in DMSO) as test substances, the test item - 4, 4’-Isopropylidenediphenol, ethoxylated, esters with fatty acids, coco, induced micronucleus formation to the test system CHO-K1 at 3-hr exposure without metabolic activation with doubling the number of micronuclei at the highest dose compared to the negative control.

At the 3-hr exposure with metabolic activation and 20-hr exposure without metabolic activation only 10 micronuclei more were created at the highest dose compared to the control. Furthermore it has to considered that cytotoxicity is 60% at 20-hr exposure without metabolic activation at the highest dose. According to the OECD guideline care should be taken in interpreting positive results only found in the higher end of this 55 ± 5% cytotoxicity range. In addition, the number of micronuclei at 20-hr exposure without metabolic activation increases inversely with the concentration and corresponds to increasing cytotoxicity with the concentration.

Therefore, based on the above study, the induction of micronucleus formation by the test item - 4, 4’-Isopropylidenediphenol, ethoxylated, esters with fatty acids, coco, to the test system CHO-K1 at 3-hr exposure with and without metabolic activation and 20-hr exposure without metabolic activation is considered not to be clearly positive as a statistically significant increase in micronucleus formation compared with the concurrent negative control cannot be exhibited. Further tests are necessary for clarification.

Executive summary:

The substance 4, 4’-Isopropylidenediphenol, ethoxylated, esters with fatty acids, coco, was tested in a study according to OECD 487.

In the study, using CHO-K1 as test system and 5.0 µl/ml, 1.7 µl/ml and 0.6 µl/ml of test item (dissolved in DMSO) as test substances at 3- hr exposure with and without S9 mixture,

a)   the number of micronuclei in the presence of negative control substance was significantly lower than that of the positive control substance;

b)   there was dose-related increase in the number of micronuclei formed by the test substance comparing with negative control. But a significance cannot be determined based on this study performance.

In the study, using CHO-K1 as test system and 5.0 µl/ml, 1.7 µl/ml and 0.6 µl/ml of test item (dissolved in DMSO) as test substances at 20- hr exposure without S9 mixture,

a)   the number of micronuclei in the presence of negative control substance was significantly lower than that of the positive control substance;

b)   there was an inverse dose-related increase in the number of micronuclei formed by the test substance comparing with negative control.

Therefore, based on the above study, using 5.0 µl/ml, 1.7 µl/ml and 0.6 µl/ml of test item (dissolved in DMSO) as test substances, the induction of micronucleus formation by the test item - 4, 4’-Isopropylidenediphenol, ethoxylated, esters with fatty acids, coco, to the test system CHO-K1 at 3-hr exposure with and without metabolic activation and 20-hr exposure without metabolic activation is considered not to be clearly positive as a statistically significant increase in micronucleus formation compared with the concurrent negative control cannot be exhibited. Further tests are necessary for clarification. 

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
[Please provide information for all of the points below. The information should be specific to the endpoint for which testing is proposed. Note that for testing proposals addressing testing on vertebrate animals under the REACH Regulation this document will be published on the ECHA website along with the third party consultation on the testing proposal(s).]

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out : 4,4'-Isopropylidenediphenol, ethoxylated, esters with fatty acids, coco

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies : In vitro mammamlian cell micronucleus study is ambiguous
- Available non-GLP studies : Not available
- Historical human data : Not available
- (Q)SAR : Not available
- In vitro methods : The conducted in vitro mammamlian cell micronucleus study is ambiguous: According to REACH Annex VIII no. 8.4.4 column 2 an appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII.
- Weight of evidence : Not available
- Grouping and read-across: Not available


CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The conducted in vitro mammalian cell micronucleus study is ambiguous: According to REACH Annex VIII no. 8.4.4 column 2 an appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed [if relevant]

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)