Phenol, paraalkylation products with C10-15 branched olefins (C12 rich) derived from propene oligomerization, carbonates, calcium salts, overbased, sulfurized, including distillates (petroleum), hydrotreated, solvent-refined, solvent-dewaxed, or catalytic dewaxed, light or heavy paraffinic C15-C50

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test - In this key 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 (Lawlor, 1997) for an analog test material 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 (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.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5th to 15th April 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study following GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100 and TA 102 & E. coli WP2 uvrA

Details on mammalian cell type (if applicable):

Species and cell type: Male Sprague-Dawley rat, liver S-9 fractionQuantity: 25 µl/plate

Additional strain / cell type characteristics:

other: Histidine-deficient strains of Salmonella typhimurium and a tryptophan-deficient strain of Escherichia coli were grown.

Metabolic activation:

with and without

Metabolic activation system:

0.5 mL of 5% v/v S-9 mix per plate Induced or not induced: Aroclor 1254 induced, 500 mg/kg i.p.

Test concentrations with justification for top dose:

0, 0.033, 0.1, 0.33, 1.0, and 3.33 mg/plate, with and without S-9

Vehicle / solvent:

The vehicle used was 25% w/w Pluronic 127 (surfactant, CAS # 9003-11-6) in ethanol.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: See table in materials and methods section below.

Details on test system and experimental conditions:

METHOD OF APPLICATION: All media and solutions were prepared as described by Maron and Ames (1983). Plate incorporation test.
Three replicate plates were evaluated for each treatment.

DURATION
The bacterial tester strain culture was grown by inoculating nutrient broth with a tester strain and incubating with shaking at ~37°C to a density of 1-2 x 10ˆ9 cells/ml. Without metabolic activation, 100 µl of tester strain and 100 µl of solvent or test material were added to 2.5 ml of molten selective top agar at ~45°C. With metabolic activation, 100 µl of tester strain, 100 µl of solvent or test material, and 0.5 ml of S-9 mix were added to 2.0 ml of molten selective top agar at ~45°C. After vortexing, the mixture was overlaid onto the surface of 25 ml of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 hours at ~37°C. The number of revertant colonies was counted and the presence and condition of the bacterial lawn was noted. Solvent and positive controls were run concurrently.

Test material was not completely miscible with the top agar at >5 mg/plate. The test material mixture for the highest dose level (10 mg/plate) was too viscous to deliver accurately.
Testing at the highest pipettable dose level (5 mg/plate) with 25-400 µl S-9/plate on strains TA98 and TA100 indicated that increasing the concentrations of S-9 did not signficantly increase the induction of revertants; therefore, 25 µl S-9/plate was used for the mutagenicity assay.

DETERMINATION OF CYTOTOXICITY
- Method: The test materials were checked for toxicity on strain TA100 with and without S-9 mix at dose levels of 0.003 to 10 mg/plate. No toxicity was observed.

Evaluation criteria:

The following criteria were met before the results of the mutagenicity assay were judged as positive or negative:
(1) the high dose showed some toxicity, or was 10 mg/plate or the limit of solubility;
(2) the spontaneous controls were within the laboratory's acceptable range;
(3) the positive controls produced at least a three-fold increase in the number of revertants over the values for the respective negative controls.

Species / strain:

other: S. typhimurium TA 98, TA 100 and TA 102 & E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

When tested at concentrations of 0.033 to 3.33 mg/plate the test material was not mutagenic to TA98, TA100, TA102, or E. Coli WP2 uvrA with or without metabolic activation provided by Aroclor-induced rat liver S-9. No cytotoxicity was observed at any concentration. The apparent increase in strain TA102 with S-9 metabolic activation is not considered biologically significant; the mean ± SEM for historical solvent control values is 325 ± 30.

The tester strains responded to the positive controls, known mutagens, as expected.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

First criterion for a valid test (as stated in the report) was not met. Dose of 5 mg/plate was the highest pipettable dose level and the solubility limit in top agar. Other test validity criteria were satisfied. No information was presented on condition of the background lawn, which would help assessment of cytotoxicity. Nevertheless, highest test dose of 3.33 mg/plate was close to the solubility limit and clearly not cytotoxic or mutagenic, based on revertants/plate data. The greatest mean number of revertants/plate over concurrent control among all test doses and all strains was 42% with S-9 and 19% without S-9. There was no indication of a dose-related decrease or increase of mean revertants/plate. Statistical results were not applicable.

Conclusions:

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

Executive summary:

In a GLP compliant study conducted in line with the standard methodology published by Ames, 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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1st April to 21st May 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Strain: 3.7.2C
Cells were cultured in Fischer's medium in a shaker incubator at 37°C in humidified 5% CO2 in air. Cultures were diluted daily to a cell density of approximately 3 x 10ˆ5 cells per ml. Each time a culture was used, it was checked for bacterial or fungal contamination.
Prior to use in the assay, L5178Y cells were treated with methotrexate to reduce the frequency of spontaneously occurring TK-/- cells.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor S-9 metabolic activation

Test concentrations with justification for top dose:

0, 75, 100, 150, 200, 250, and 275 µg/mL with S-9
0, 60, 70, 80, 90, 100, and 110 µg/mL without S-9

Vehicle / solvent:

5% Pluronic F-68 (surfactant, CAS # 9003-11-6) in water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Migrated to IUCLID6: with S-9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Three replicate plates were evaluated for each treatment.

DURATION
- Test Material Exposure: Based on the data derived from the toxicity test, the test material was prepared so that the highest concentration would yield a percent total growth of approximately 10%. The test material was solubilized and diluted to produce evenly spaced dose levels which would yield approximately 90% total growth at the lowest dose. The test material was added to cells, both with and without metabolic activation and incubated for approximately 4 hours. Cells were then washed and placed into suspension culture at a density of 0.3 x 10ˆ6 cells/mL.

- Expression Time: In order for induced mutations to be expressed, the cells must undergo several divisions. This period is designated as the expression time. After the initial exposure to the test material the cells were incubated for 2 days and adjusted to 0.3 x 10ˆ6 cells/mL at 24 hours.

- Cloning: At the end of the expression period, a portion of the cells were plated in
(a) restrictive medium containing trifluorothymidine (TFT) which allowed only the TK-/- cells to grow, and
(b) non-restrictive medium which indicated cell viability.
The plates were incubated at 37°C in humidified 5% CO2 in air for 10-12 days.

- Accumulation of Data: After the incubation period, both the mutagenicity (TFT restrictive medium) plates and the viability (non-restrictive medium) plates were scored for the total number of colonies per plate using an Artek 880 colony counter or by hand. The frequency of mutants induced by each test material dose was determined by comparing the average number of colonies in the mutagenicity plates to the average number of colonies in the corresponding viability plates. Induced mutagenic activity, if any, was quantified by comparing the mutant frequency of the treated plates to that of the control plates.

DETERMINATION OF CYTOTOXICITY
The test material was checked for toxicity with and without S-9 metabolic activation at dose levels of 10 ug/ml to 5000 µg/ml. Toxicity was observed at 100, 500, 1000, 2500 and 5000 ug/ml with and without S-9.

Evaluation criteria:

Criteria for an Acceptable Mouse Lymphoma Screen: All the following criteria should be met for the mutagenicity screen to be considered valid:
- The mutant frequency of the appropriate positive control (either with or without S-9 activation) should be at least twice that of the appropriate negative (solvent) control.
- The spontaneous mutant frequency of the negative (solvents control should be in the range of 0.2 to 2.0 per 10ˆ4 cells.
- The plating efficiency of the negative (solvent) control should be at or above 50%.
- The test material should be tested to the level of approximately 10% total growth, or the limits of solubility, or to a high dose of 10 mg/ml. Test materials may be tested as slurries.

The following criteria must be met for the outcome of the Mouse Lymphoma Screen (either with or without S-9 metabolic activation) to be considered mutagenic:
The mutant frequency of one or more test material concentrations, with >10% total growth, is at least two times greater than that of the negative (solvent) control.

The following result must be met for the outcome of the Mouse Lymphoma Screen (either the with or without metabolic activation) to be considered non-mutagenic:
None of the mutant frequencies of any of the test material concen-trations, with a survival of >10% total growth, are at least two times greater than that of the solvent control.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>100 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results of the preliminary Toxicity Test (not shown) conducted on the test material indicated significant toxicity at 100 µg/ml for the cultures with and without S-9 metabolic activation. Based on the results of the Toxicity Test, the test material was tested in the Mutagenicity Screen at concentrations ranging from 10 µg/ml to 275 µg/ml with metabolic activation and from 30 µg/ml to 110 µg/ml without activation. After the two-day expression period, six cultures with metabolic activation and six cultures without activation were selected for cloning based on the degree of observed toxicity. The cultures were cloned at 75, 100, 150, 200, 250, and 275 µg/ml (with metabolic activation) and 60, 70, 80, 90, 100, and 110 µg/ml (without activation).

None of the cultures treated with test material with or without metabolic activation exhibited mutant frequencies significantly different from the average mutant frequencies of the corresponding negative, solvent controls. Percent total growth ranged from 32% to 94% (with metabolic activation) and 10% to 95% (without activation). The positive control (DMBA) responded satisfactorily.

Under the conditions tested the test material was not mutagenic to L5178Y TK+/- cells in this assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

First criterion for a valid test (as stated in the report) was not met without S-9, total growth only 32%. Total cell growth at the high dose without S-9 was 10%. Other test validity criteria were satisfied. The highest mutation frequency over control among all test doses was 4% with S-9 and 51% without S-9. There was no indication of a dose-related increase of mutation frequency with S-9. There was a trend without S-9 to higher mutation rates with progressively lower total growth at doses of 80-110 µg/mL. No statistical analysis was performed on the study results, as they were not needed.

Conclusions:

Under the conditions tested the test material was not mutagenic to L5178Y TK+/- cells in this assay.

Executive summary:

In a study conducted in line with OECD guideline 476 and under conditions of GLP, 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) 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

Study period:

11th September to 11th October 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed per recognized guideline following GLP. This study is being used as read across from EC 701-249-4, therefore reliability is reduced to 2.

Justification for type of information:

See read across/category assessment and justification attached in section 13 for more information

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Crl:CD-1®(ICR) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI.
- Age at study initiation: All animals were eight weeks and three days old at the time of dosing.
- Weight at study initiation: The weight range of the animals used in the micronucleus assay was 23.7-35.1 and 21.2-28.2 grams for the males and females, respectively.
- Assigned to test groups randomly: Yes
- Housing: Animals were housed five per cage during quarantine, and housed at least five per cage at randomization. Sanitized caging was used for housing the animals.
- Diet/water (e.g. ad libitum): A commercial diet (Purina® Certified Laboratory Pellets ® # 5002) and water were available ad libitum for the duration of the study. The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water was analyzed on a retrospective basis for specified microorganisms, pesticides, alkalinity, heavy metals, and halogens.
- Acclimation period: Animals were quarantined for seven days before being placed on study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 6 °F
- Humidity (%): 55 ± 15%
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained

Route of administration:

intraperitoneal

Vehicle:

The vehicle control, peanut oil (Sigma, Lot # 83H0848), was administered concurrently with the test article at a volume of 10 mL/kg.

Details on exposure:

Dosing suspensions were prepared just prior to dosing and were prepared by making a 500 mg/mL stock for the high dose (5000 mg/kg ). This was prepared by adding peanut oil (Sigma, Lot # 83H0848) to the test material resulting in a dark brown viscous solution. Dilutions of this stock were prepared for the 2500 and 1250 mg/kg and dose levels. Volumes dosed were 10 mL/kg and were based upon individual animal weights. All dosing stocks were continuously mixed during the dosing procedure.

Duration of treatment / exposure:

The animals dosed with the test article and the vehicle control were euthanized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow.

Frequency of treatment:

Once intraperitoneally

Post exposure period:

24, 48 and 72 hours post-dose

Remarks:

Doses / Concentrations:
0, 1250, 2500 and 5000 mg/kg b.wt.
Basis:
nominal conc.
The dose volume was 10 mL/kg b.wt. for all groups.

No. of animals per sex per dose:

Five mice/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide at a dose of 60 mg/kg b.wt.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on results from the dose selection study, dose levels of 1250, 2500 and 5000 mg/kg were selected for testing in this study.

DETAILS OF SLIDE PREPARATION:
At the appropriate harvest time, the animals were euthanized by CO2/O2 inhalation followed by penetration of the thorax. A limb bone was removed from each hind leg and the adhering soft tissue and epiphyses were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3 - 5 mL bovine serum (one tube for each animal). Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, and stained in May-Grunwald solution followed by Giemsa (Schmid, 1975). The air-dried slides were coverslipped.

METHOD OF ANALYSIS:
The slides were coded for analysis, and scored for micronuclei and the polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this Crl:CD-1®(ICR) BR strain is about 0.0 - 0.4%.

The frequency of PCEs versus NCEs was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring at least the first 1000 erythrocytes.

Evaluation criteria:

The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ringshaped micronuclei occasionally occurred. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).

Statistics:

Results for each sex / harvest time were analyzed by ANOVA on either untransformed (when variance homogeneous) or rank transformed (when
variance heterogeneous) micronucleus cell count data. If significance was observed with ANOVA, a Dunnett’s t-test was used to determine which
dose groups were different from the negative control. A Cochran-Armitage test for linear trend was used to evaluate dose-response.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Dose levels of 1625, 2750, 3875 and 5000 mg/kg were administered by intraperitoneal injection for the dose selection study.
- Clinical signs of toxicity in test animals:
All animals were examined after dosing and daily throughout the duration of the study (three days) for toxic effects and/or mortalities. All animals appeared normal immediately after dosing and remained healthy until the end of the observation period. No mortality occured.
- Rationale for exposure: Based on these results, the maximum tolerated dose was estimated to be >5000 mg/kg.


RESULTS OF DEFINITIVE STUDY
All animals were observed immediately after dosing and periodically throughout the duration of the assay for toxic symptoms and/or mortalities. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest times. All test article dosed groups appeared normal immediately after dosing and remained healthy until the appropriate harvest times.
The test article induced no statistically significant increases in micro-nucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The test article did not induce a statistically significant change in the PCE:NCE ratio. The positive control, Cyclophosphamide, induced statistically significant increases in micronucleated PCEs in both sexes as compared to the vehicle controls, with means and standard errors of 2.38% ± 0.54% and 4.28% ± 0.79% for the males and females, respectively.

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.

Executive summary:

In a study conducted in accordance with GLP to OECDguideline 474, based on the results of the dose selection study, the maximum tolerated dose was estimated as >5000 mg/kg. In the micronucleus assay, the test article was suspended in peanut oil and dosed by intraperitoneal injection at 1250, 2500 and 5000 mg/kg. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. The animals dosed with the test article and the vehicle control were euthanized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow. The animals dosed with the positive control were euthanized approximately 24 hours after dosing for extraction of the bone marrow.

One thousand PCEs per animal were scored. The test article did not induce a statistically significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test. The test article did not induce a statistically significant change in the PCE:NCE ratio.

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.

There was no available data to fulfil this endpoint for the test material and so the study was read-across from a supporting substance (EC.701 -249 -4)

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 EC 701-251-5 did not test all the strains per current guidelines. Therefore, an OECD 471 study with an analog substance, EC 701-249-4 is also provided. The difference between the substances is that 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 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 target and analogs, no classification is warranted.