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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1980/07/24-1980/12/08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on structural analogue suitable for read across. This non-GLP study was conducted similar to OECD guideline and has significant deviations to warrant restriction.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report date:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only 4/5 recommended strains of bacteria tested and no information regarding cytotoxicity
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-Pr) esters, zinc salts
EC Number:
272-723-1
EC Name:
Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-Pr) esters, zinc salts
Cas Number:
68909-93-3
Molecular formula:
Too complex
IUPAC Name:
Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-Pr) esters, zinc salts
Details on test material:
Test material (EC 272-723-1) from lot EC24321 was described as brown or golden yellow, viscous liquid. The test article was stored in glass in the dark at room temperature.
Justifications for read-across was present in section “ANY OTHER INFORMATION ON RESULTS INCL. TABLES”

Method

Target gene:
Point (reverse) mutations in selected histidine requiring mutant strains of Salmonella typhimurium. Tester strains have a deletion through uvrB to remove DNA excision repair function and rfa to remove the lipopolysaccharide coat. TA1535 is a missense mutant which is reverted by agents which cause base pair substitutions. TA98, TA1538, TA1537 are frame shift mutants. TA98 is TA1538 and TA100 is TA1535 with the additions of pKM101 plasmid.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
1.0, 0.3, 0.1, 0.03 and 0.01 ul/plate
Vehicle / solvent:
All dilutions were made with ethyl acetate due to solubility and lack of reactivity with test article.
Controlsopen allclose all
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 -S9 Migrated to IUCLID6: 10 ug/plate
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 and TA 1538 -S9 Migrated to IUCLID6: 5 ug/plate
Untreated negative controls:
yes
Remarks:
bacteria only
Negative solvent / vehicle controls:
yes
Remarks:
ethyl acetate
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene 5 ug/plate
Remarks:
For all strains +S9
Details on test system and experimental conditions:
The assay was performed by plate incorporation and the method of Ames et al with modification. The assay was performed in duplicate, with (+S9) or without (-S9) metabolic activation. Each of the tester strains was dosed with 5 concentrations of test substance, solvent controls, and a positive control. Three plates/dose group/strain/treatment set were evaluated. 100 ul of test substance, positive control or vehicle control were added to each plate along with 100 ul of tester strain, S9 mix (if required) and 2.0 ml of molten top agar. This was overlaid onto the surface of bottom agar in a petri dish and plates were incubated for 48 h at 37 C. The number of revertant colonies were counted on a Biotran II automatic colony counter.
Evaluation criteria:
All sterility controls must be negative for bacterial growth. The average number of revertant colonies representing spontaneous mutation must be within the following acceptable range: TA1535, 6-60; TA1537, 3-16; TA1538, 6-35; TA100, 80-250; TA98, 10-60. The average number of revertant colonies in the positive control plates must be at least 5 times those of the solvent control.

Statistics:
Mean revertant colony count and standard deciation were calculated for each dose level and compared to solvent controls.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Remarks:
not addressed in original submission
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Remarks:
not addressed in original submission
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A range finder was not included in this report.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

JUSTIFICATIONS FOR READ-ACROSS

AMES assay is not available for EC 270-608-0, however experimental data on structurally related substances EC 283-392-8 and EC 272-723-1 are available and suitable for read-across. Justifications for read-across:

Manufacture/Usage:

EC 270-608-0, EC 283-392-8, and EC 272-723-1 are generically referred to as zinc dialkylthiophosphate (ZDDP) which are produced under similar manufacturing procedures and used in commerce as multi-functional anti-wear and anti-oxidation inhibitor performance components in passenger motor oils, diesel engine oils and industrial oils such as hydraulic lubricants.

Chemical Similarity:

EC 270-608-0, EC 283-392-8, and EC 272-723-1 consist of alkyl substituted phosphorodithioic acid structures complexed with zinc. These three ZDDPs share similar core structures - alcohol ester of dithiophosphate, specific structural variations that relate to their alcohol group substituent are the alkyl chain length and the degree of branching of the alcohol charge.

EC 270-608-0:Phosphorodithioic acid, mixed O,O-bis(iso-butyl and pentyl) esters, zinc salts, referred to as “mixed isobutyl and pentyl derivative”

EC 283-392-8:phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-propyl) esters, zinc salts, referred to as “mixed isopropyl and 1,3-dimethylbutyl derivative”

EC 272-723-1:Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-propyl) esters, zinc salts, referred to as “mixed 2-ethylhexyl and isopropyl derivative”.

Using Tanimoto Fingerprint (ToxMatch Version 1.06 software) to model chemical structures of the analogues showed comparable values for relevant molecular descriptors (e.g., number of H bond acceptor atoms), and gave a similarity index greater than 0.8 (values range from 0, no similarity to 1, identical; peer reviewed literature indicates that values greater than 0.6 are significantly similar); therefore chemical structures of the analogues have determined to be sufficiently close for there to be a reasonable expectation of similar toxicological effects.

Physicochemical Properties:

Standard physicochemical properties for each substance are shown below:

Table 1. Establishment of similarity between the data donating substance and the data accepting substance

EC

Alkyl group

MW

log Kow

Water Sol (ppm)

283-392-8

(Donate data)

C3/C6, branched

576

0.56

2736

270-608-0

(Accept data)

C4, branched

C5, mixture of linear and branched

576

0.69

1625

272-723-1 

(Donate data)      

C3/C8 branched

632

0.84

2113

As shown in Table 1, these substances have similar MW, Log Kow, and they all water soluble. The similarity of the physicochemical properties of these substances parallels their structural similarity.

Biologically Active Functional Groups:

The ester group presents in each of the analogue members, and is expected to exhibit similar biological activities. Non-random patterns were observed for the toxicological effects (e.g. available data showed low levels of acute toxicity effect, consistent trend of change in ecotoxicity effect, etc.), these common behaviors and consistent trends suggest a common mechanism/mode of action, with little influence from the length of carbon chain. These facts further supported read-across between the analogue members.

Available Data and Adequacy for Read-across:

EC 283-392-8:negative inassay with S. typhimurium TA 1535, TA 1537 TA 98 and TA 100 and E. coli WP2 uvrA (with and without metabolic activation).

EC272-723-1: negative inassay with S. typhimurium TA 1535, TA 1537, TA 1538 TA 98 and TA 100 (with and without metabolic activation).

By read across to these two studies, EC 270-608-0 was predicted to be not mutagenic in bacteria.

Conclusion:

In vitro bacterial gene mutation assays have been conducted for EC 283-392-8 and EC 272-723-1. Frequencies of reverse mutations in bacteria were not significantly changed after exposure to these substances. The findings in bacterial mutation assay did not vary in proportion to the alkyl chain length. Based on the abovementioned justifications, results from EC 272-723-1 and EC 283-392-8 were weighted and used to fill this data gap for EC 270-608-0. EC 270-608-0 was predicted to be non-mutagenic in bacteria.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

No detectable mutagenic activity for EC 272-723-1 was found under the conditions of this study. This result was used to fill this data gap for EC 270-608-0. EC 270-608-0 was predicted to be non-mutagenic in bacteria.
Executive summary:

In a bacterial reverse mutation assay in the presence and absence of exogenous metabolic activation, Salmonella strains TA1535, TA1537, TA1538, TA98, and TA100 were treated with test substance at concentrations of 0.03, 0.01, 0.006, 0.003, 0.001 ul/plate. The negative and positive controls fulfilled the requirements for determination of a valid test. None of the test substance treatment conditions produced a statistically significant increase in the number of revertants. Based on the results of this study, the test substance would not be classified based on weight of evidence for the chemical class in accordance with the classification system of GHS.

This toxicity study is classified as acceptable and satisfies the guideline requirement for in vitro mutagenicity test.