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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The results of an Ames test and in vitro metaphase analysis in human lymphocytes in vitro using a mixture containing 30.2% zinc 3,5-bis(alpha-methylbenzyl)salicylate, were negative. The result of an in vitro mammalian cell gene forward mutation test at the hgprt locus of the Chinese hamster ovary using zinc 3,5-bis(α-methylbenzyl)salicylate was negative.
Link to relevant study records
Reference
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:
October 2012 - March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study, no restrictions
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 treated rat liver S9 fraction
Test concentrations with justification for top dose:
Expt 1: 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL
Expt 2: 187.5, 375, 750, 1500, 3000 and 5000 µg/mL
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Ethyl Methanesulfonate (0.4 µL/mL) was used -S9; and Benzo(a)pyrene (6 µg/mL) was used +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1 x 10^6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test chemical is considered positive in the mutation assay if:
i. A positive result is defined as a concentration-related biologically significant increase in mutant frequency in comparison with concurrent negative control and the test item causes a three-fold increase (Nestmann, E.R. et al., 1991) in the number of 6-thioguanine resistant colonies relative to concurrent negative control and such increases are statistically significant and outside of the laboratory historical negative (solvent used) control range.
ii. A net increase in mutant colonies of treated above the concurrent control is observed in at least two of the concentrations tested.

Negative results were confirmed by a repeat test (short duration), using a modification in test concentrations.
Statistics:
Weighted regression analysis was performed to evaluate the dose response relationship (Li, A.P. et al., 1987; Hsie, A.W. et al., 1981) on zinc 3,5-bis(α-methylbenzyl)salicylate treatment groups against negative control (excluding positive controls).
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Absolute Cloning Efficiency at Selection and Mutant Frequency

The mean absolute cloning efficiency (ACE) at selection and mean mutation frequency per 1 x 106cells (MF) in the absence and presence of metabolic activation for trial I and trial II are provided below:

 

 

Group

(µg/mL)

TRIAL I

 

Group

(µg/mL)

TRIAL II

- S9

+ S9 (2% v/v S9)

- S9

+ S9 (2% v/v S9)

Mean ACE

Mean MF

Mean ACE

Mean
MF

Mean ACE

Mean
MF

Mean ACE

Mean
MF

NC (DMSO)

0.8286

9.36

0.7570

9.57

NC (DMSO)

0.7830

13.31

0.7562

16.03

T1 (156.25)

0.7811

8.54

0.7411

8.70

T1 (187.5)

0.7403

15.85

0.7482

12.28

T2 (312.5)

0.7227

10.31

0.7279

12.93

T2 (375)

0.7215

13.88

0.7331

15.27

T3 (625)

0.6721

13.44

0.6706

14.60

T3 (750)

0.7170

14.53

0.7510

13.20

T4 (1250)

0.6813

12.45

0.7243

9.23

T4 (1500)

0.6970

14.59

0.6879

15.91

T5 (2500)

0.6740

9.64

0.6188

10.85

T5 (3000)

0.6780

12.55

0.6299

16.14

T6 (5000)

0.4964

13.53

0.5339

17.26

T6 (5000)

0.5248

14.65

0.5249

17.06

PC

0.5282

221.88

0.5351

221.46

PC

0.5391

236.03

0.5536

201.95

 

Conclusions:
Interpretation of results (migrated information):
negative

From these results, it is concluded that zinc 3,5-bis(α-methylbenzyl)salicylate does not have potential to induce gene mutations at the hgprt locus of CHO-K1 cells, both in the absence and presence of metabolic activation under the present experimental conditions.
Executive summary:

The mutagenic potential of zinc 3,5-bis(α-methylbenzyl)salicylate was assessed both in the absence and presence of metabolic activation using the CHO-K1 cell line. Zinc 3,5-bis(α-methylbenzyl)salicylate was tested in two independent experiments, with and without metabolic activation. Ethyl methanesulfonate (0.4 µL/mL) and benzo(a)pyrene (6 µg/mL) were used as the positive control in the absence and presence of metabolic activation, respectively.

The CHO-K1 cells were exposed to zinc 3,5-bis(α-methylbenzyl)salicylate at the test concentrations of ranging from 156.25, to 5000 µg/mL both in the absence and presence of metabolic activation for a period of 4 hours.

No significant dose-related effect was observed at any of the treated concentrations during either trial of the experiments.

No relevant influence of the test item on pH value or osmolality was observed. The spontaneous mutation level was (negative control) within the acceptable limit in both the trials, validating the acceptability of the test system. The increased mutant frequency observed in positive controls demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

From these results, it is concluded that zinc 3,5-bis(α-methylbenzyl)salicylate does not have potential to induce gene mutations at thehgprtlocus of CHO-K1 cells both in the absence and presence of metabolic activation under the present experimental conditions. 

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

Additional information

Additional information from genetic toxicity in vitro:

In vitro mammalian cell gene forward mutation test at the hgprt locus of the Chinese hamster ovary (CHO)-K1 cell line using zinc 3,5-bis(α-methylbenzyl)salicylate: From these results, it is concluded that zinc 3,5-bis(α-methylbenzyl)salicylate does not have potential to induce gene mutations at the hgprt locus of CHO-K1 cells, both in the absence and presence of metabolic activation under the present experimental conditions.

Mixture containing 30.2% zinc 3,5-bis(alpha-methylbenzyl)salicylate: Reverse Mutation Assay ''Ames Test'' Using Salmonella Typhimurium Project Number 318/38: The mixture was found to be non-mutagenic under the conditions of this test.

Mixture containing 30.2% zinc 3,5-bis(alpha-methylbenzyl)salicylate: Metaphase Analysis In Human Lymphocytes In Vitro Project Number 318/39: The mixture produced no significant increases in the frequency of chromosome aberrations either in the presence or absence of a liver enzyme metabolising system; this mixture is therefore considered to be non-clastogenic to human lymphocytes in vitro.

Justification for read-across

Two of the three studies described above were conducted using a liquid mixture containing 30.2% zinc 3,5-bis(alpha-methylbenzyl)salicylate.

The remaining substances present in the mixture include:

-         5.5% PSMS-11

-         1.1% PVA

-         0.7% DIBLAME

-         < 1% Other

-         Remainder: water

PSMS-11 and PVA are not considered to be hazardous substances. DIBLAME is classified as H315: Causes skin irritation, H317: May cause an allergic skin reaction and H411: Toxic to aquatic life with long lasting effects. However, the concentration of DIBLAME in the mixture is below that which triggers classification of the mixture for these hazards. Therefore, it is considered justified to read-across from this mixture to zinc 3,5-bis(α-methylbenzyl)salicylate.


Justification for selection of genetic toxicity endpoint
Only study categorised as Klimisch 1.

Justification for classification or non-classification

The above studies have been ranked reliability 1 or 2 according to the Klimisch et al system. This ranking was deemed appropriate because these studies are conducted according to recognised test methods. Sufficient dose ranges and numbers are detailed; hence they are appropriate for use based on reliability and animal welfare grounds.

The above results triggered no classification in accordance with the CLP Regulation (EC No 1272/2008).