Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-Pr) esters, zinc salts

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted on structural analogue and suitable for read across.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts

EC Number:

283-392-8

EC Name:

Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts

Cas Number:

84605-29-8

IUPAC Name:

Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: 8 weeks and 2 days
- Weight at study initiation: 28.9-36.4 g (male) and 23.1-29.0 g (female)
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: five per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 15
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Peanut oil
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: 10 mL/kg
- Amount of vehicle (if gavage or dermal): 10 mL
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required):
- Purity:

Duration of treatment / exposure:

24, 48, and 72 hours

Frequency of treatment:

Once

Post exposure period:

24, 48, and 72 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
7.13 mg/kg
Basis:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
14.3 mg/kg
Basis:

Doses / concentrations 3

Remarks:

Doses / Concentrations:
28.5 mg/kg
Basis:

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: sterile water
- Doses / concentrations: 60 mg/kg

Examinations

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Range finding study performed to find the maximum tolerated dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):


DETAILS OF SLIDE PREPARATION:
Slides fixed with methanol and stained in May-Grunwald solution followed by Giemsa.

METHOD OF ANALYSIS:
Scored for micronuclei and the polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) cell ration.

OTHER:

Evaluation criteria:

Statistically sifnificant dose-related increase in micronucleated PCE's and the detection of a statictically sifnificant postive response for at least one dose level.

Statistics:

The frequency of micronucleated polychromatic erythrocytes between treated groups and vehicle controls were compared. Tests included Cochran-Armitage test for trend, a one-way analysis of variance and Dunnett’s procedure.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

JUSTIFICATIONS FOR READ-ACROSS

Chromosome aberration effects (in vivo) were not evaluated for EC 272-723-1, however experimental data on structurally related substances EC 283-392-8 and EC 224-235-5 were available and suitable for read-across. Justifications for read-across:

Manufacture/Usage:

EC 272-723-1, EC 283-392-8 and EC 224-235-5 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 272-723-1, EC 283-392-8, and EC 224-235-5 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 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”.

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 224-235-5:Phosphorodithioic acid, O,O-bis(2-ethylhexyl) ester, zinc salt, referred to as “2-ethylhexyl 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 were listed in Table 1. 

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

EC	Alkyl group	Average MW	Log Kow	Water Sol (ppm)
283-392-8 (data source)	C3/C6, branched	576	0.56	2764
272-723-1  (accept data)	C3/C8 branched	632	0.84	2111
224-235-5 (data source)	C8 branched	772	3.6	8.8

 

As shown in Table 1, the physicochemical data reflect a consistent trend according to the order of MW: as MW increases, Log Kow increases whereas water solubility decreases. Therefore, these substances were determined to be similar and/ follow a regular, predictable pattern for biological effects. 

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, lacking of mutagenic effect in bacteria, 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 in chromosome-aberration (mammalian erythrocyte micronucleus assay) in male and female rats.

EC 224-235-5:negative in chromosome-aberration (mammalian erythrocyte micronucleus assay) in male and female rats.

These studies were reliable and adequate for read-across.

Conclusion:

Mammalian erythrocyte micronucleus assay has been conducted for EC 283-392-8 and EC 224-235-5. The frequency of micronucleated polychromatic erythrocytes between treated groups and vehicle controls were not significantly changed after exposure to these substances. The findings in chromosome-aberration did not vary correspondingly to the alkyl chain or physicochemical differences. Based on the abovementioned justifications, results from these analogous substances were weighted and used to fill this data gap for EC 272-723-1. EC 272-723-1 was predicted to be non-cryptogenic in rats.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Mammalian erythrocyte micronucleus assay has been conducted for EC 283-392-8 and EC 224-235-5. The frequency of micronucleated polychromatic erythrocytes between treated groups and vehicle controls were not significantly changed after exposure to these substances. The findings in chromosome-aberration did not vary correspondingly to the alkyl chain or physicochemical differences. Based on the abovementioned justifications, results from these analogous substances were weighted and used to fill this data gap for EC 272-723-1. EC 272-723-1 was predicted to be non-cryptogenic in rats.