Reaction mass of (Z)-3-(9-octadecenyloxy)propane-1,2-diol and alpha-(Z)-octadec-9-en-1-yl-omega-hydroxy-di[oxy[(hydroxymethyl)-1,2-ethanediyl]] and alpha-(Z)-octadec-9-en-1-yl-omega-hydroxy-tri[oxy[(hydroxymethyl)-1,2-ethanediyl]]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 February - 31 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

9022-76-8

Details on test material:

- Name of test material (as cited in study report): Chimexane NB
- Physical state: Yellow to brown viscous liquid
- Analytical purity: 98.5 % active substance
- Lot/batch No.: 0129336
- Expiration date of the lot/batch: February 2007
- Storage condition of test material: Room temperature, protected from light exposition (coloration may occur)
- Stability: Stable

Method

Target gene:

His+ for S. typhimurium

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: see table 7.6.1/1

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 fraction prepared from liver homogenates of Sprague-Dawley OFA male rats induced with Aroclor 1254 (500 mg/kg bw) by intraperitoneal route

Test concentrations with justification for top dose:

PRELIMINARY CYTOTOXICITY ASSAY
- With and without S9 mix: 50, 150, 500, 1500 and 5000 μg/plate in TA 1535, TA 1537, TA 98, TA 100 and TA 102 using plate-incorporation method.

MUTAGENICITY ASSAYS
- Experiment 1 (without S9 mix, plate-incorporation method): 50, 150, 500, 1500, 3000 and 5000 μg/plate (TA 1535); 15, 50, 150, 500, 1500 and 3000 μg/plate (TA 1537); 15, 50, 150, 500 and 1000 μg/plate (TA 98); 0.5, 1.5, 5, 15, 50 and 150 μg/plate (TA 100 and TA 102)
- Experiment 1 (with S9 mix, plate-incorporation method): 50, 150, 500, 1500 and 5000 μg/plate (TA 1535, TA 1537, TA 98 and TA 102); 1.5, 5, 15, 50 and 150 μg/plate (TA 100)
- Experiment 2 (without S9 mix, plate-incorporation method): 50, 150, 500, 1000 and 2000 μg/plate (TA 1535 and TA 1537); 15, 50, 150, 500 and 1000 μg/plate (TA 98); 1.5, 5, 15, 50 and 100 μg/plate (TA 100 and TA 102)
- Experiment 2 (with S9 mix, pre-incubation method): 50, 150, 500, 1500 and 5000 μg/plate (TA 1535, TA 1537, TA 98 and TA 102); 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 100)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide (1 µg/plate for TA 100 and TA 1535); 9-Aminoacridine (50 µg/plate for TA 1537); 2-Nitrofluorene (2 µg/plate for TA 98), Mitomycin C (0.125 µg/plate for TA 102)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine: 2 and 1 µg/plate for without and with pre-incubation, respectively for TA 1537, TA 1535, TA 98 and TA 100; Benzo[a]pyrene: 2 µg/plate for without and with pre-incubation for TA 102

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: All five strains were obtained from B.N. Ames, University of California, Berkeley, U.S.A.

METHOD OF APPLICATION: Plate incorporation and preincubation methods

DURATION:
- Pre-incubation period for 60 minutes at 37 °C
- Incubation period for plates: 48-72 h at 37 °C (preliminary cytotoxicity assay) 48 h at 37 °C (mutagenicity assay)

NUMBER OF REPLICATIONS:
- Preliminary cytotoxicity assay: 1 plate/dose
- Mutagenicity assays: 3 plates/dose


DETERMINATION OF CYTOTOXICITY:
- Method: Cytotoxicity was checked by microscopic examination of the background lawn of plates.

OTHERS:
- Sterility of the test item, media and S9 mix were tested.
- Reading of results: Colonies were counted using colony counter. The results were expressed as the mean number of mutants per plate and, for each concentration of the test product, the following ratio was established:
Mean number of revertants per plate in presence of the test item / Mean number of revertants per plate without the test item

Evaluation criteria:

Criteria based on biological significance:
- Strains TA 1535 and TA 1537: A 3-fold increase in the number of revertants compared to the vehicle control, at any dose level and/or evidence of a dose relationship was considered as a positive result in the assay.
- Strains TA 98, TA 100 and TA102: A 2-fold increase in the number of revertants compared to the vehicle control, at any dose level and/or evidence of a dose relationship was considered as a positive result in the assay.

Criteria based on statistical significance:
- Data were analysed by means of Dunnett's method (Mahon et al, 1989) allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control.

Reproducibility:
- A positive result observed in a single assay that could not be reproduced in at least 2 independent assays could not be considered of biological significance.

- All these criteria are not absolute, but they, however, help in coming to a decision, which can be conclusive in the majority of the cases (Brusick, 1980).

Comparison to historical control data:
- In some borderline cases, an additional criterion to be considered is the comparison between the number of revertants induced by the test compound and the laboratory historical control data. Indeed, an increase in each individual value that is above the highest value of corresponding historical control data can help supporting a conclusion such as “equivocal” or “weak” mutagen.

Statistics:

Data were analysed by means of Dunnett's method (Mahon et al, 1989) allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Solubility: Test item was not soluble in distilled water but it was highly soluble in DMSO up to 50 mg/mL.
Precipitation:
Preliminary cytotoxicity assay:
- Slight precipitation of the test substance on the plates was observed at 5000 μg/plate in all five strains.
Mutagenicity assays:
- Experiment 1: Slight precipitation was observed at 150 μg/plate (TA 100 and TA 102, -S9), 1000 μg/plate (TA 98, -S9) and 3000 μg/plate (TA 1537, -S9).
- Experiment 2: No precipitation was observed at any of the doses tested in any of the strains.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Historical control data of the solvent and positive controls of the year 2005 were used to compare the revertant frequencies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Preliminary cytotoxicity assay:
Without S9 mix:
- In strain TA 1537, at the highest dose of 5000 µg/plate, the test item induced a slight toxicity characterized by a decrease in the number of revertants.
In strain TA 98, the test item was found toxic, characterized by a total absence of revertants at the doses of 1500 and 5000 µg/plate.
- Test item was strongly toxic in both strains TA 100 and TA 102, at the highest dose (5000 µg/plate) where total absence of growth was noted. A strong toxicity was observed at the doses of 150, 500 and 1500 µg/plate, where a strong to slight toxicity on the background lawn and a total absence of revertants were noted.

With S9 mix:
- Slight toxicity (TA 1537, TA 98 and TA 102) was observed at 5000 µg/plate. Slight toxicity on the background lawn at 5000 µg/plate and strong decrease in the number of revertants from 50 to 5000 µg/plate was observed in TA 100.

Mutagenicity assays: Statistically significant decreases in the number of revertants were observed during the first and the second assay, both with and without metabolic activation, in all strains, particularly at the highest tested doses.

Without S9 mix:
- Experiment 1: Slight to moderate toxicity as well as a strong decrease in the number of revertants were observed at the dose of 5000 µg/plate in TA 1535, of 3000 µg/plate in TA 1537, of 150 µg/plate in TA 100 and TA 102. Cytotoxicity was observed at 1000 µg/plate in TA98.
- Experiment 2: Slight to moderate toxicity as well as a strong decrease in the number of revertants was observed at the doses of 1000 and 2000 µg/plate in strains TA 1535 and TA 1537, at the highest dose of 1000 µg/plate in strain TA98, and at 100 µg/plate in strains TA 102 and TA 100

With S9 mix:
- Experiment 1: Cytotoxicity was observed at 5000 μg/plate in TA 102.
- Experiment 2: Cytotoxicity was observed at 150, 500, 1500 and 5000 μg/plate (TA 100) and 5000 μg/plate (TA 102).

OTHERS:
- Sterility test of test item, S9 mix and media: No contamination was observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/2: Mean revertant frequencies

Strains	Doses (µg/plate)	Mean revertants per plate
		Experiment 1				Experiment 2
		-S9		+S9#		-S9		+S9##
		Mean	R	Mean	R	Mean	R	Mean	R
TA 1535	Solvent control	13.5	-	8.5	-	10.7	-	5.8	-
	50	20.7	1.5	12.3	1.4	11.3	1.1	10.3	1.8
	150	19.0	1.4	10.0	1.2	7.0	0.7	7.0	1.2
	500	15.0	1.1	7.7	0.9	3.7	0.3	5.3	0.9
	1000	-	-	-	-	2.3*	0.2	-	-
	1500	7.0	0.5	5.3	0.6	-	-	1.7*	0.3
	2000	-	-	-	-	0.3*	0	-	-
	3000	2.0*	0.1	-	-	-	-	-	-
	5000	0.3*	0	4.3	0.5	-	-	2.3	0.4
	PC	477.3*	40.8	572*	59	363.3*	36.3	162.3*	25.8
TA 1537	Solvent control	5.0	-	2.83	-	2.8	-	3.7	-
	15	2.7	0.5	-	-	-	-	-	-
	50	2.0	0.4	3.3	1.16	2.7	1	4.7	1.3
	150	2.7	0.5	5.0	1.76	3.3	1.2	5.0	1.4
	500	3.0	0.6	2.7	0.95	2.3	0.8	2.5	0.7
	1000	-	-	-	-	1.7	0.6	-	-
	1500	2.7	0.5	2.7	0.95	-	-	2.7	0.7
	2000	-	-	-	-	0.0*	0	-	-
	3000	1.0	0.2	-	-	-	-	-	-
	5000	-	-	3.7	1.31	-	-	1.3	0.4
	PC	418.3	126.8	361.3*	84	452.0*	150.7	111.0*	22.2
TA 98	Solvent control	18.2	-	20.2	-	18.3	-	16.8	-
	15	15.7	0.9	-	-	16.7	0.9	-	-
	50	13.3	0.7	24.7	1.2	15.0	0.8	16.7	1
	150	13.7	0.8	20.7	1	9.7	0.5	21.0	1.3
	500	11.3	0.6	29.0	1.4	6.7	0.4	14.7	0.9
	1000	9.3	0.5	-	-	5.0*	0.3	-	-
	1500	-	-	21.0	1	-	-	21.7	1.3
	5000	-	-	24.0	1.2	-	-	19.0	1.1
	PC	1101.3*	60.2	2698.7*	118.9	711.3*	35.6	1354.7*	81.1
TA 100	Solvent control	115.2	-	95.0	-	110.3	-	88.8	-
	0.5	111.0	1	-	-	-	-	-	-
	1.5	118.0	1	105.7	1.1	81.3	0.7	108.7	1.2
	5	107.0	0.9	107.0	1.1	90.3	0.8	96.7	1.1
	15	113.3	1	91.7	1	77.0	0.7	93.3	1.1
	50	87.7	0.8	109.0	1.1	64.0*	0.6	94.0	1.1
	100	-	-	-	-	22.3*	0.2	-	-
	150	48.7*	0.4	101.3	1.1	-	-	84.3	0.9
	500	-	-	-	-	-	-	49.7*	0.6
	1500	-	-	-	-	-	-	0.0	0
	5000	-	-	-	-	-	-	0.0	0
	PC	625.3*	4.9	3904*	44.4	412.0*	3.1	858.7*	10.3
TA 102	Solvent control	182.5	-	257.0	-	223.7	-	271.5	-
	0.5	221.3	1.2	-	-	-	-	-	-
	1.5	263.0*	1.4	-	-	197.3	0.9	-	-
	5	226.3	1.2	-	-	198.0	0.9	-	-
	15	189.3	1	-	-	209.7	0.9	-	-
	50	222.7	1.2	304.7	1.2	174.7	0.8	258.7	1
	100	-	-	-	-	79.3*	0.4	-	-
	150	118.3*	0.6	279.7	1.1	-	-	283.3	1
	500	-	-	281.0	1.1	-	-	238.7	0.9
	1500	-	-	261.3	1	-	-	259.0	1
	5000	-	-	216.3	0.8	-	-	179.3*	0.7
	PC	1525.7*	6	1082.7*	4.2	1468.3*	6.4	1517.0*	5.9

 

#:without pre-incubation; ##: with pre-incubation; R: ratio = number of mutants in the treated / number of mutants in the control; *: p<0.01 (Dunnett's t)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under these test conditions, Chimexane NB is not mutagenic with and without metabolic activation in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 according to the Annex VI of the Directive 67/548/EEC and the Regulation (EC) N° 1272-2008 (CLP).

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) was exposed to Chimexane NB at the following concentrations:

Preliminary cytotoxicity assay:

- With and without S9 mix: 50, 150, 500, 1500 and 5000 μg/plate in TA 1535, TA 1537, TA 98, TA 100 and TA 102 using plate-incorporation method.

Mutagenicity assays:

- Experiment 1 (without S9 mix, plate-incorporation method): 50, 150, 500, 1500, 3000 and 5000 μg/plate (TA 1535); 15, 50, 150, 500, 1500 and 3000 μg/plate (TA 1537); 15, 50, 150, 500 and 1000 μg/plate (TA 98); 0.5, 1.5, 5, 15, 50 and 150 μg/plate (TA 100 and TA 102)

- Experiment 1 (with S9 mix, plate-incorporation method): 50, 150, 500, 1500 and 5000 μg/plate (TA 1535, TA 1537, TA 98 and TA 102); 1.5, 5, 15, 50 and 150 μg/plate (TA 100)

- Experiment 2 (without S9 mix, plate-incorporation method): 50, 150, 500, 1000 and 2000 μg/plate (TA 1535 and TA 1537); 15, 50, 150, 500 and 1000 μg/plate (TA 98); 1.5, 5, 15, 50 and 100 μg/plate (TA 100 and TA 102)

- Experiment 2 (with S9 mix, pre-incubation method): 50, 150, 500, 1500 and 5000 μg/plate (TA 1535, TA 1537, TA 98 and TA 102); 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (TA 100)

Metabolic activation system used in this study was 10 % S9 mix. S9 fraction was prepared from liver homogenates of Sprague-Dawley OFA male rats induced with Aroclor 1254 (500 mg/kg bw) by intraperitoneal route. Vehicle and positive control groups were also included in this test.

In preliminary cytotoxicity assay, slight precipitation was observed in the plates at 5000 μg/plate in all five strains. Test substance induced toxicity at 150, 500, 1500 and 5000 μg/plate (TA 100 and TA 102, -S9), 5000 μg/plate (TA 1537, -S9; TA 1537, TA 98, TA 100 and TA 102, +S9). Statistically significant decreases in the number of revertants were observed during the first and the second assay, both with and without metabolic activation in all strains. The positive and vehicle controls induced the appropriate responses in the corresponding strains. Test item showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations either in the absence or presence of S9 mix.

Under these test conditions, Chimexane NB is not mutagenic with and without metabolic activation in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 according to the Annex VI of the Directive 67/548/EEC and the Regulation (EC) N° 1272-2008 (CLP).