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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test - In the first key analog (EC 701 -251 -5) study for in vitro genetic toxicity (Machado et al, 1985, report number: SOCAL 2321) there was no guideline specified, however it was considered to be comparable to OECD Guideline 471 (Bacterial Reverse Mutation Assay). The study was conducted in line with GLP. A reliability rating of 1 according to the criteria of Klimisch, 1997.

The test material was tested in the histidine-deficient strains of Salmonella typhimurium TA98, TA100, and TA102 and in the tryptophan-deficient strain of Escherichia coli WP2 uvrA at dose levels of 0.033 to 3.33 mg/plate with and without metabolic activation provided by Aroclor-induced rat liver S-9. The test material was suspended in 25% Pluronic 127 (w/w in ethanol). The suspension was miscible with the top agar. It was not cytotoxic at any concentration tested.

 

Under the conditions tested, the test material was not mutagenic to TA98, TA100, TA102, or E. coli WP2 uvrA.

 

An additional Ames tests is included as a second key study for a different analog test material (EC 701 -249 -4) (Lawlor, 1997) to cover additional strains not tested in the key study (Machado et al., 1985).

 

Mouse lymphoma- In this key study for in vitro genetic toxicity with an analog (EC 701 -251 -5) (Winingeret al, 1985, report number: SOCAL 2322) there was no guideline specified, however it was considered to be comparable to OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test). The study was conducted in line with GLP. A reliability rating of 1 according to the criteria of Klimisch, 1997.

The test material was tested in the L5178Y TK+/- Mutagenicity Screen with and without S-9 metabolic activation. The cultures with activation were tested at concentrations ranging from 75 μg/ml to 275 μg/ml; cultures without activation were tested at concentrations ranging from 60 μg/ml to 110 μg/ml.

 

The results indicated that the test material did not induce a significant increase in the mutant frequencies of cultures tested either with or without metabolic activation. Under the conditions tested the test material was not mutagenic.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In the key study for in vivo genetic toxicity (Ivett, 1997, Corning Hazleton report number: 17865-0-455CO) with an analog substance (EC 701 -249 -4), the study was conducted according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test). The study was conducted in line with GLP.

 

The reliability rating for this study is 1, however this is being used as read across from a supporting substance (EC 701-249-4) as there was no available data to fulfil this endpoint for the test material and so the reliability rating will be reduced to 2, according to the criteria of Klimisch, 1997.

Based on the results of the dose selection study, the maximum tolerated dose was estimated as >5000 mg/kg.

The test article did not induce a statistically significant increase in micro-nuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse micronucleus assay.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
See category justification attached in Section 13 for more information. Evaluating alkyl phenate sulfides (“phenates”) as a category is appropriate based on similar chemical structures and starting materials, manufacturing processes, physical and chemical properties, functional uses as a lubricating oil additive, and toxicological data. Regarding the ECHA Read-Across Assessment Framework (2017), the alkyl phenate category fit into Scenario 6 (different compounds with the same effect and no variation in the strength of that effect across substances).

Phenates in this category are manufactured in a similar way and from the same staring alkylphenol, tetrapropenyl phenol (“TPP”, EC 310-154-3; AKA phenol, dodecyl-, branched (PDB, PDDP)). The primary difference among the phenates is if they have calcium carbonate basing; the amount of overbasing may also differ. However, based on the trends observed with the robust toxicology data for the category, the amount of calcium carbonate overbasing is not expected to alter the hazards, especially as the core phenate functionality does not change and calcium carbonate has a low hazard potential.

Based on the data and consistent trends observed among category members, phenates have low hazard for human health and the environment. The registered (target) substance, EC 701-208-0 is very similar to EC 701-251-5 and this substance can be used as direct read across (and is used as the test material in the target record, where data exists for this source substance); EC 701-249-4 and 272-388-1 serve to bracket EC 701-208-0 with EC 701-251-5 regarding different levels of calcium carbonate overbasing.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

There was no mortality and all animals appeared normal without sign of adverse effect until sacrifice. Mean percentage of micronucleated PCEs was within the range of laboratory historical controls for all treatment and vehicle control groups.

Conclusions:
The test article did not induce a statistically significant increase in micro-nuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse micronucleus assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The alkyl phenate sulfide category has multiple negative OECD 471 studies. The study with analog substance EC 701-251-5 did not test all the strains per current guidelines. However, an OECD 471 study with another simialr substance, EC 701-249-4, did. The difference between the alkyl phenate sulfides is that the registered substance (EC 701 -208 -0) and EC 701-251-5 has calcium carbonate overbasing whereas EC 701-249-4 has calcium dihydroxide. However, this difference does not change the toxicology as explained in the read across and category assessment attached in section 13. This includes that neither calcium hydroxide nor calcium carbonate are genotoxic (Health Council of Netherlands, 2003; EU, 2008; ECHA BPC, 2016).

In addition, a study with EC 272-388-1 was also negative in an OECD 471 study (OECD SIAR, 2009). EC 272-388-1 does not have calcium hydroxide added and provides additional evidence that the alkyl phenate sulfide molecule is not mutagenic.

No in vivo genotoxicity data is available for the registered substance (EC701 -208 -0) or EC 701-251-5. Therefore, the OECD 474 study with EC 701-249-4 is used to fill this gap.

To further evaluate the genotoxicity potential as well as justify the read across, OASIS TIMES v.2.27.15.146 was used to predict whether chromosomal aberrations may occur. The suitability of OASIS TIMES was reviewed in a JRC Scientific and Technical Report (Serafimova, Gatnik, and Worth, 2010) where it was found to have advantages over other predictive models for genotoxicity as it incorporates metabolism.

To start, UVCB G Graph 1.0 was used to create a Generic SMILES to incorporate all variations possible in the UVCB nature of the registered substance. Over 1500 isomers were predicted and a filter was used to reduce the number to a manageable but representative amount of structures to be predicted by TIMES. The filter option selected was Molecular Weight with 3 intervals in order to separate the isomers based on the amount of sulfur bridging (1-3). Five members were randomly selected from each distribution group for a total of 15 constituents for final modeling. 

The difference between the registered substance and EC 701-249-4 is the degree of calcium carbonate overbasing. Based on the principles in “Specific Rules of Ionic Characteristics of Metal Salts of Organic Chemicals” found in the OASIS software, dissociation of the carbonate group is likely to occur after exposure based on the large differences in electronegativity resulting from the ionic bond between the calcium and oxygen ions. Based on the principle that dissociation will occur at the Ca-O bond, both the registered substance and read-across substance would be expected to dissociate to the same phenol, thiobis substance. Therefore, TIMES was modeled with and without hydrolysis selected to mimic dissociation and non-dissociation structures, respectively.

Running the model without dissociation (calcium carbonate present) resulted in negative predictions for the parent structures. However, the corresponding parent structures modeled with the calcium dissociated yielded metabolites that were positive in situ for chromosomal aberration due to aromatic ring hydroxylation and the potential formation of hydroquinones and catechols and subsequent oxidation to benzoquinones. The difference is likely due to the calcium carbonate preventing transformation to a quinone species. 

All of the predicted positive metabolites are below 0.05 for probability to obtain. Therefore, the likelihood that the metabolites would be formed and available to interact with DNA is very low. This is consistent with the negative micronucleus study for EC 701-249-4 as transformation to genotoxic species does not appear to occur in an animal model. 

As stated, TIMES predicted negative results for both parents and metabolites when hydrolysis was turned off (with calcium carbonate) supporting that genotoxicity is not expected for the target substance. This illustrates that dissociation of the calcium carbonate is required for transformation to genotoxic metabolites in situ. Therefore, read across may be considered conservative, although the primary evidence from the QSAR predictions is that read across is appropriate and alkyl phenate sulfides are not genotoxic.

References:

1.      Health Council of the Netherlands. Health-based reassessment of administrative occupational exposure limits for calcium carbonate (CAS 471-34-1). 2003.

2.      European Union (EU). Draft Assessment Report for calcium carbonate. Volume 1. August, 2008.

3.      ECHA Biocidal Products Committee (BPC). Opinion on the application for approval of hydrated lime. April 14, 2016. 

Justification for classification or non-classification

Based on multiple negative genotoxicity studies for the alkyl phenate sulfide category, no classification is warranted.