[No public or meaningful name is available]

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:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

OECD guideline compliant as at 1987

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

as at 1987

Deviations:

yes

Remarks:

only 4 strains tested (E. Coli not tested), only plate incorporation assay used.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty alcohols C13-15 (odd numbered, linear and branched), reaction products with ethylene oxide, sodium chloroacetate and ethanolamine

Molecular formula:

R-O-(CH2CH2O)nCH2CO-NH-CH2CH2OH

IUPAC Name:

Fatty alcohols C13-15 (odd numbered, linear and branched), reaction products with ethylene oxide, sodium chloroacetate and ethanolamine

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): AA 15
- Physical state: yellow viscous liquid

Method

Target gene:

TA1535: his G46 rfa- delta uvrB-
TA1537: his C3076 rfa- delta uvrB-
TA98: his D3052 rfa- delta uvrB- R+
TA100: his G46 rfa- delta uvrB- R+

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

Plate incorporation assay up to 10,000 µg Fe/plate + and - S9. Pre-incubation assay

Vehicle / solvent:

Vehicle used: distilled water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene for all strains

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours concurrent with exposure

SELECTION AGENT (mutation assays): minimal agar

NUMBER OF REPLICATIONS: doses were tested in triplicate in the plate incorporation assay . 5 dose levels were tested.

DETERMINATION OF CYTOTOXICITY
- Method:
• one Salmonella strain (TA98), based on the assumption that all strains used show a similar toxic response with and wo activation.
• concentrations: 0, 1.6, 8, 40, 200, 1000 and 5000 µg/plate.
• analysis of appearance of a complete bacterial lawn as seen under a dissecting microscope after 24 hours incubation at 37°C.

Evaluation criteria:

A statistically significant increase in the mean number of revertants that exceeds twice the concurrent negative control, plus evidence of a dose response relationship.

Statistics:

• analysis of variance on each set of data to obtain the F-statistic
• in case of statistical significance (p<0.05) and dose related increases correlation coefficient is calculated for the response range and the significance of the result is determined from standard tables.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A pKM 101

Remarks:

E. coli WP2 uvrA, not used in this study

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Remarks:

AMINOL A15 LA 438-7 (FB-4136)

Positive controls validity:

not applicable

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: at 5000 µg/plate with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: at 5000 µg/plate with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: at 5000 µg/plate with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: at 5000 µg/plate with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

GENOTOXIC EFFECTS:

- The test item caused restricted growth of the background lawns for all four strains, both with and without S-9, at 5000 µg/plate.

- No dose related increases in revertant rates were seen with any of the strains except for TA1537 and TA98 without activation at the highest dose. The effects occur only at the highest dose and are deemed rather survivors of the cytotoxic effect than mutants. In addition the effects are neither dose related nor reproducible in experiment 2 and therefore not regarded as a positive mutagenic response.

- High concentrations of the test item caused decreases in numbers of revertants (except for the two concentrations nentioned above).

- All solvent (negative) controls gave counts of revertants within normal ranges

- All positive controls gave numbers of induced revertants within expected ranges demonstrating the sensitivity of the assay and the metabolising activity of the S-9 mix.

PRECIPITATION CONCENTRATION: None reported

Table 1: Pretest

Strain	% S-9	Concentration of Test Substance (µg/plate)*
		0	1.6	8	40	200	1000	5000
TA98	0	+	+	+	+	+	+	+
TA98	10	+	+	+	+	+	+	+

* These are actual amounts of test substance added and correspond to solutions of test compound with concentrations from 16 µg/ml to 50 mg/ml.

+ = restricted growth

+ = normal growth

Table 2: MEAN NUMBER OF REVERTANTS PER PLATE FOR THE TEST ITEM

Strain	% S-9	Concentration of Test Substance (µg/plate)
		0	8	40	200	1000	5000	PC
TA1535	0	9.0	13.0	9.0	6.7	6.0	1.7	322.3
TA1537	0	3.0	5.0	3.7	4.0	1.7	1.7	1013.0
TA98	0	10.7	11.7	5.3	5.3	4.7	3.7	173.3
TA100	0	93.0	88.3	80.7	77.0	72.0	57.3	287.3
TA1535	10	11.0	12.3	7.7	10.7	7.0	4.7	95.7
TA1537	10	5.7	3.3	6.0	5.0	3.3	3.3	35.7
TA98	10	16.0	13.7	11.3	9,7	11.0	9.0	523.0
TA100	10	92.0	86.0	89.7	80.3	72.7	72.0	639.7

PC = Positive control

Table3: MEAN NUMBER OF REVERTANTS PER PLATE FOR THE TEST ITEM

Strain	% S-9	Concentration of test substance (µg/plate)
		0	8	40	200	1000	5000	PC
TA1535	0	5.3	8.3	4.7	6.3	4.7	1.7(a)	262.3
TA1537	0	6.3	7.0	5.7	5.3	2.7	58.3(a)	925.7
TA98	0	10.0	13.7	10.0	9.0	9.0	25.0(a)	138.0
TA100	0	112.0	106.7	81.3	84.0	67.3	58.0(a)	218.3
TA1535	10	10.0	9.7	8.7	6.7	4.3	3.7(a)	186.7
TA1537	10	6.7	4.3	4.3	4.3	2.3	3.3(a)	34.0
TA98	10	13.0	8.7	14.7	10.3	4.3	5.3(a)	138.3
TA100	10	104.0	94.7	81.7	89.7	72.7	67.7(a)	369.3

PC = Positive control

a = Reduced background lawn

Applicant's summary and conclusion

Conclusions:

Under the test conditions, test substance is not mutagenic with and without metabolic activation in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, up to 5000 µg/plate.

Executive summary:

The test item was tested in vitro by the Ames plate incorporation method for its ability to induce mutations in four histidine dependent auxotrophic mutants of Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 according to OECD 471 as at 1987.

Two independent mutation tests were performed, each in both the presence and absence of a metabolic activation system (S-9 mix) derived from the livers of Aroclor 1254 treated rats. The bacteria were exposed to the test material dissolved in Dimethylsulphoxide, which was also the solvent control. Based on the results of a preliminary toxicity rangefinder, 5000 µg/plate of AA 15 was chosen as the highest dose level to be used, with lower dose levels of 1000, 200, 40 and 8 µg/plate.

All solvent (negative) controls gave counts of revertants within normal ranges.

All positive controls gave numbers of induced revertants within expected ranges demonstrating the sensitivity of the assay and the metabolising activity of the S-9 mix.

The test item produced no significant increases in the number of revertants, with any of the tester strains, in either of the two experiments performed in both the presence and absence of metabolic activation, when assayed up to 5000 µg/plate.

Under the test conditions, test substance is not mutagenic in Salmonella typhimurium strains with and without metabolic activation, up to 5000 µg/plate.