Santicizer 2148

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-01-30 to 1990-06-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Escherichia coli strain not tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of dodecyldiphenyl phosphate and tetradecyldiphenyl phosphate

Molecular formula:

CH24H35O4P.C26H39O4P

IUPAC Name:

Reaction mass of dodecyldiphenyl phosphate and tetradecyldiphenyl phosphate

Test material form:

liquid

Specific details on test material used for the study:

Purity: 90-95%
Lot no: DEEUGEA NBP#4321285
EHL Test sample T900009
Stated expiration date: January 1991
Storage conditions: Room Temperature

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
Salmonella typhimurium strains TA1535 were obtained from the Laboratory of Dr. B. N. Ames (Berkeley, California).

Additional strain / cell type characteristics:

other: Proper phenotype of each culture was verified for cyrstal violet sensitivity, ampicillin resistance, requirement for histidine and biotin, and spontaneous reversion frequency.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
Salmonella typhimurium strains TA1537 were obtained from the Laboratory of Dr. B. N. Ames (Berkeley, California).

Additional strain / cell type characteristics:

other: Proper phenotype of each culture was verified for cyrstal violet sensitivity, ampicillin resistance, requirement for histidine and biotin, and spontaneous reversion frequency.

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
Salmonella typhimurium strains TA98, were obtained from the Laboratory of Dr. B. N. Ames (Berkeley, California).

Additional strain / cell type characteristics:

other: Proper phenotype of each culture was verified for cyrstal violet sensitivity, ampicillin resistance, requirement for histidine and biotin, and spontaneous reversion frequency.

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
Salmonella typhimurium strains TA100, were obtained from the Laboratory of Dr. B. N. Ames (Berkeley, California).

Additional strain / cell type characteristics:

other: Proper phenotype of each culture was verified for cyrstal violet sensitivity, ampicillin resistance, requirement for histidine and biotin, and spontaneous reversion frequency.

Metabolic activation:

with and without

Metabolic activation system:

- source of S9 : S9 preparation was purchased from Molecular Toxicology Inc. (College Park, Maryland)
- method of preparation of S9 mix : Prepared from livers of Aroclor-1254 induced male Sprague-Dawley rats (Hilltop Laboratories, Scottsdale, PA). Prepared using procedure described by Ames et al. 1975.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 was tested for metabolic activation capability in a matrix experiment in which both percent S9 in S9 mix and the amount of positive standard per plate were varied.

Test concentrations with justification for top dose:

0.03, 0.10, 0.30, 1.00, and 3.00 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: anhydrous acetone

- Justification for choice of solvent/vehicle: Not specified

- Justification for percentage of solvent in the final culture medium: Not specified

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

9-aminoacridine

benzo(a)pyrene

other: Sodium Nitrite (-S9: TA1535); 2-aminoanthracene (+S9: TA1535 and TA1537)

Details on test system and experimental conditions:

General procedures were basically those described by Ames et al.

Doses used per plate with/without S-9 were 0.1, 0.3, 1.0, 3.0, and 5.0 mg (Toxicity Screen). Based on the toxicity and insolubilty observed in the toxicity screen, the maximum dose level for mutagenicity testing was selected as 3 mg/plate.

Plate incorporation tests were performed by mixing 0.1 ml of bacterial culture, and. if appropriate. 0.5 ml of S-9 mix with 2 ml of histidine-biotin top agar (0.5% (w/v) NaCl, 0.6% (w/v) Difco agar, 0.05 mM L-histidine-HCl, 0.05 mM biotin) maintained at 44-48 degrees C. THe mixture was poured onto minimal glucose agar plates (Vogel-Bonner medium E with 2% glucose and 1.5% Difco agar). Toxicity tests employed the same procedures as those used in the plate incorporation test. Single plates were prepared for each strain/S-9/dose level combination for the toxicity test. 3 replicate plates were prepared for each strain/S-9/combination for the plate incorporation tests. Concurrent positive and negative controls were conducted for plate incorporation tests to demonstrate strain sensitivity and metabolic activation system capability. Plates were examined after at least 48 hours at 37 degrees Cl. A screen for suitable solvents, for the possibility of pH or osmolality effects, and for reaction with plastic petri dishes was performed prior to the plate incorporation tests.

Revertant colonies for plates with more than 500 revertant colonies/plate were estimated by counting revertant colonies in several fields under a stereomicroscope and multiplying the counted colonies by a factor related the total plate area to the area of the counted fields. Revertant colonies measured in this manner are calculated to not more than three significant figures. Revertant colonies on other plates, except as noted, were counted with an Artek Model 880 automatic colony counter or counted by visual examination (<10 revertants/plate).

Evaluation criteria:

Results were considered to be clearly positive for a strain/microsome combination if revertants/plate values were significantly elevated over control values (p<0.01) at three treatment levels, and there was a statistically significant dose response (p<0.01).

Statistics:

Statistical analysis was performed on plate incorporation assay results after transforming revertants/plate values as log10(revertants/plate). Analysis included Bartlett's test for homogeneity of variance and comparison of treatrments with controls using within-levels pooled variance and a one-sided t-test. Grant's test was performed to determine if outliers were present. Statistical significance of dose response was evaluated by regression analysis for log 10 transformed values and revertants plate.

A critical level of p<0.01 was used to determine statistical significance. Results with P<0.05 were also indicated to assist in interpretation of results.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Toxicity Test Results with test strain TA100

Amount of test material per plate (mg)	S-9*	Toxic Response**	Solubility†
0.1	-	N	S
0.1	+	N	S
0.3	-	N	S
0.3	+	N	S
1.0	-	N	S
1.0	+	N	S
3.0	-	T	I
3.0	+	T	I
5.0	-	T	I
5.0	+	T	I

* = S-9 Mix was prepared using 10% (v/v) rat liver S-9 preparation (MolTox 02337) in the S-9 Mix

** = N = No toxic response; T = Toxicity observed

† = S = Test material soluble; I = Test material insoluble

 

Table 2. Statistical summary of plate incorporation test results for TA98 and TA100 with and without S9

Strain	TA98	TA98	TA100	TA100
Activation System	With S-9	None	With S-9	None
Test Date	09-Feb-90	09-Feb-90	09-Feb-90	02-Mar-90
Amount/plate (mg)	Revertants/plate Mean and standard deviation in ( )
0.03	38.0 (± 8.3)	27.7 (± 2.1)	93.0 (± 11.5)	123 (± 6.2)
0.10	34 (± 7.5)	28.7 (± 9.1)	103.7 (± 3.1)	114.3 (± 13.0)
0.30	34.7 (± 4.0)	24.0 (± 3.5)	95.7 (± 9.1)	124.0 (± 13.2)
1.00	36.0 (± 5.3)	25.7 (± 4.5)	93.3 (± 7.5)	110.7 (± 20.0)
3.00 (T, I) †	35.0 (± 4.0)	20.5 (± 3.5)	0.0 (± 0)	111.5 (± 4.9)
Solvent Controls	33.7 (± 6.8)	26.0 (± 2.3)	98.0 (± 10.6)	123.0 (± 10.4)
Summary Analysis
Treatment levels with Rev/Plate > Control
p<0.06	0	0	0	0
p<0.01	0	0	0	0
Bartlett’s test	N	N	N	N
No. of outliers (Grubb’s test)	0	0	0	0
Dose Response	N	N	N	N
Lack of fit test	N	N	N	N

ll analyses performed with Log(10) transformed data.

Codes used are: * = significant at p<=0.06 level; ** = significant at p<=0.01 level; N = Not significant at p<=0.06 level

† = T = Toxicity observed, I = Test material insoluble

Table 3. Statistical summary of plate incorporation test results for TA1535 and TA1537 with and without S9

Strain	TA1535	TA1535	TA1537	TA1537
Activation System	With S-9	None	With S-9	None
Test Date	13-Feb-90	13-Feb-90	13-Feb-90	13-Feb-90
Amount/plate (mg)	Revertants/plate Mean and standard deviation in ( )
0.03	11.7 (± 3.2)	10.7(± 2.5)	12.0 (± 1.7)	9.3 (± 3.1)
0.10	11.0(±1.0)	9.0 (± 1.0)	13.3 (± 5.0)	9.7 (± 6.4)
0.30	10.3(± 1.2)	8.7 (± 3.1)	12.3 (± 2.1)	6.7 (± 1.5)
1.00	11.7(± 3.2)	11.3 (± 0.6)	11.0 (± 3.6)	6.3 (± 0.6)
3.00 (T, I) †	12.0 (± 1.0)	6.0 (± 1.4)	11.3 (± 1.6)	7.7 (± 2.1)
Solvent Controls	11.9 (± 3.1)	14.3 (± 1.8)	9.7 (± 2.6)	7.2 (± 2.3)
Summary Analysis
Treatment levels with Rev/Plate > Control
p<0.06	0	0	0	0
p<0.01	0	0	0	0
Bartlett’s test	N	N	N	N
No. of outliers (Grubb’s test)	0	0	0	0
Dose Response	N	N	N	N
Lack of fit test	N	A	N	N

ll analyses performed with Log(10) transformed data.

Codes used are: * = significant at p<=0.06 level; ** = significant at p<=0.01 level; N = Not significant at p<=0.06 level; A = Data do not allow lack-of-fit test to be performed

† = T = Toxicity observed, I = Test material insoluble

Table 4. Statistical summary of plate incorporation test results for TA98 and TA100 with and without S-9

Strain	TA98	TA98	TA100	TA100
Activation System	With S-9	None	With S-9	None
Test Date	23-Feb-90	21-Jun-90	23-Feb-90	23-Feb-90
Amount/plate (mg)	Revertants/plate Mean and standard deviation in ( )
0.03	40.0 (± 6.8)	19.0(± 6.6)	88.3 (± 17.8)	96.0 (± 22.9)
0.10	35.0(± 6.6)	17.6 (± 0.7)	94.0 (± 16.6)	99.3 (± 22.1)
0.30	36.6(± 7.0)	18.3 (± 6.8)	93.7 (± 0.6)	109.7 (± 12.5)
1.00	37.3(± 7.0)	14.7 (± 4.2)	89.7 (± 11.6)	(T)115.0 (± 18.7) †
3.00 (T, I) †	0.0 (± 0.0)	17.7 (± 0.6)	0.0 (± 0.0)	107.3 (± 8.4)
Solvent Controls	37.9 (± 0.0)	17.3 (± 6.3)	119.4 (± 14.3)	112.2 (± 20.4)
Summary Analysis
Treatment levels with Rev/Plate > Control
p<0.06	0	0	0	0
p<0.01	0	0	0	0
Bartlett’s test	N	N	*	N
No. of outliers (Grubb’s test)	0	0	A	0
Dose Response	N	N	A	N
Lack of fit test	N	N	A	N

ll analyses performed with Log(10) transformed data.

Codes used are: * = significant at p<=0.06 level; ** = significant at p<=0.01 level; N = Not significant at p<=0.06 level; A = Data do not allow lack-of-fit test to be performed

† = T = Toxicity observed, I = Test material insoluble

 

Table 5. Statistical summary of plate incorporation test results for TA1535 and TA1537

Strain	TA1535	TA1535	TA1537	TA1537
Activation System	With S-9	None	With S-9	None
Test Date	02-Mar-90	02-Mar-90	02-Mar-90	02-Mar-90
Amount/plate (mg)	Revertants/plate Mean and standard deviation in ( )
0.03	14.47 (± 1.6)	16.3(± 1.5)	11.3 (± 2.1)	12.0 (± 3.0)
0.10	14.3(± 4.6)	13.3 (± 4.0)	10.3 (± 3.2)	12.3 (± 2.1)
0.30	10.7 (± 1.6)	14.3 (± 4.2)	10.3 (± 1.6)	9.3 (± 3.1)
1.00	16.7(± 3.1)*	12.7 (± 2.1)	11.7 (± 1.0)	8.7 (± 0.0)
3.00 (T, I) †	14.0 (± 0.0)	0.0 (± 0.0)	0.0 (± 0.0)	6.0 (± 0.0)
Solvent Controls	13.0 (± 2.9)	16.0 (± 2.7)	14.7 (± 4.2)	9.9 (± 2.3)
Summary Analysis
Treatment levels with Rev/Plate > Control
p<0.06	1	0	0	0
p<0.01	0	0	0	0
Bartlett’s test	N	N	N	N
No. of outliers (Grubb’s test)	0	0	0	0
Dose Response	N	N	N	N
Lack of fit test	N	N	N	N

ll analyses performed with Log(10) transformed data.

Codes used are: * = significant at p<=0.06 level; ** = significant at p<=0.01 level; N = Not significant at p<=0.06 level

† = T = Toxicity observed, I = Test material insoluble

Applicant's summary and conclusion

Conclusions:

The test sample, XP 2563, was concluded not to be mutagenic towards any of the Salmonella typhimurium test strains used (TA98, TA100, TA1535, and TA1537) in the presence or absence of an Aroclor 1254-induced rat liver homogenate metabolic activation system (S-9 Mix)

Executive summary:

The test material, XP 2563, was tested in Ames/Salmonella plate incorporation assays using test strains TA98, TAlOO, TA1535 and TA1537 in the presence and absence of an Aroclor 1254-induced rat liver homogenate (S-9) . In the toxicity screen, toxicity and insolubility was observed at levels of 3 and 5 mg/plate with and without activation. The maximum dose level for mutagenicity testing was selected to be 3 mg/plate. No significant mutagenicity was observed in both the initial assays and the subsequent confirmation assays. Results therefore suggest that XP 2563 is not a mutagen in Salmonella typhimurium under our experimental conditions.