Didodecyl 3,3'-thiodipropionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471 (Ames test): negative

OECD 476 (in vitro mammalian cell gene mutation assay): negative

OECD 473 (in vitro mammalian chromosomal aberration test): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-08-05, 1992-11-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, performed under GLP (exposure period of 2 hours)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

(exposure period of 2 hours)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1984

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A medium, 10% (v/v) foetal calf serum (FCS), and 100 µg/mL gentamycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

29.4, 42, and 60 µg/mL (experiment I) and 60, 92.3, and 142 µg/mL (experiment II)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 2 h (with metabolic activation, cells were then washed twice with sterile saline and re-fed with fresh medium containing foetal calf serum and gentamycin, cultures were then incubated for a further 18 or 42 hours before harvesting), 20 and 44 hours (without metabolic activation)
- Expression time (cells in growth medium): 18 and 42 hours (with S9-mix), 20 and 44 hours (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 and 44 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine (one and a half hours prior to harvest, colchicine was added to give a final concentration of approximately 1 µg/mL to arrest dividing cells in metaphase)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 (4, only for the negative control)
NUMBER OF CELLS EVALUATED: 100 metaphases of each culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Determination of hyperdiploid cells: yes

Evaluation criteria:

The CHO assay was to be considered valid if the following criteria were met:
1) the binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures.
2) the proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within the normal range and the percentage of polyploid/ endoreduplicated/ hyperdiploid cells was <10%
3) at least 160 cells out of an intended 200 were analysable at each treatment level.
4) the positive control chemicals induced statistically significant increases in the number of cells with structural aberrations.

The test chemical was to be considered as clearly positive in this assay if:
1) statistically significant increases in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentration
2) the proportion of aberrant cells at such data points exceeded the normal range.
3) the results were confirmed in the second experiment.

Increases in numbers of cells with gaps or increases in the proportions of cells with structural aberrations not exceeding the normal range or occurring only at very high or very toxic concentrations were likely to be concluded as "equivocal". Cells with exchange aberrations or cells with greater than one aberration were to be considered of particular biological significance. A positive result only at the delayed harvest in Experiment II was to be taken as evidence of clastogenicity provided criteria 1 and 2 were met.

Statistics:

Aberrant cells in each culture were categorized as follows:
1. cells with structural aberrations including gaps
2. cells with structural aberrations excluding gaps
3. polyploid, endoreduplicated or hyperdiploid cells.
The totals for category 2 in negative control cultures was used to determine whether the assay was acceptable or not.
The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportion of cells in category 2 for each test treatment condition, were compared with the proportion in negative controls using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant. The proportions of cells in categories 1 and 3 were examined in relation to historical control (normal) ranges.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: low
- Precipitation: at 39 µg/mL and above

COMPARISON WITH HISTORICAL CONTROL DATA: Comparable. Few values (mostly positive controls, one test substance, and two solvent controls) exceeded historical negative control ranges.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table: Chromosomal aberrations in CHO cells after treatment with the test substance:

Experiment I
Test item	Concentration	Mitotic Index	Cells with aberrations
	in µg/mL	mean	including gaps	excluding gaps
Exposure period 2h, fixation time 18h, with S9 mix
Solvent	0	10.6	8	5
CPA	12.5	-	24	21
Test substance	29.4	14.5	6	6
	42	11.9	7	5
	60	12.8	4	3
Exposure period 20h, without S9 mix
Solvent	0	5	4	2
NQO	0.0625	-	24	23
Test substance	29.4	3.9	8	4
	42	5.1	8	4
	60	3.7	4	2
Experiment II
Exposure period 2h, fixation time 18h, with S9 mix
Solvent	0	7.3	2	2
CPA	25	-	47	46
Test substance	60	6.3	4	2
	92.3	7.3	6	4
	142	6.5	7	5
Exposure period 20h, without S9 mix
Solvent	0	4.6	5	1
NQO	0.125	-	22	22
Test substance	60	4.3	6	3
	92.3	2.6	5	3
	142	3	4	1
Exposure period 2h, fixation time 42h, with S9 mix
Solvent	0	1.8	11	6
Test substance	92.3	1.1	9	7
Exposure period 44h, without S9 mix
Solvent	0	5.2	8	2
Test substance	142	7.8	7	2

CPA: cyclophosphamide

NQO: 4-nitroquinoline-N-oxide

Conclusions:

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

Reference 2

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:

1992-08-05, 1992-10-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

preceding the most recent guideline (adopted in 1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

tk locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 plus 0, 10, or 20% horse serum (heat inactivated) plus Gentamycin and Fungizone and Pluronic (absent in RPMI 1640 plus 20% horse serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

31.25, 62.5, 125, and 250 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: 0.05 and 0.10 µg/mL (in DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 2.00 and 3.00 µg/mL (in DMSO)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT) (3 µg/mL)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The assay was considered valid if the following criteria were met:
1) the mutant frequencies in the negative (solvent) control cultures fell within the normal range (not more than 3 times the historical mean value)
2) at least 1 concentration of each of the positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies is greater than half the historical mean value).
The test substance was considered to be mutagenic if:
1) the assay was valid
2) the mutant frequency at 1 or more doses was significantly greater than that of the negative control
3) there was a significant dose-relationship as indicated by the linear trend analysis
4) the effects described above were reproducible.

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment dose, and secondly the data were checked for a linear trend in mutant frequency with treatment dose. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Survival rate: 102.9 % and 114.4 % in the absence and presence of S-9 respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Survival rate: 95.8 % and 101.5 % in the absence and presence of S-9, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: None
- Water solubility: Low
- Precipitation: at 125 and 250 µg/mL

RANGE-FINDING/SCREENING STUDIES: 2-fold intervals and ranging from 7.813 to 250 µg/mL. No marked toxicity was observed at any dose in the range-finder experiment. Precipitation was observed at the top 2 doses (125 and 250 µg/mL) both in the absence and presence of S-9 but precipitation was not evident at the end of the 3 hour incubation period. No marked toxicity was observed at any dose in the range-finder experiment.

COMPARISON WITH HISTORICAL CONTROL DATA: Within range

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: (experiment 1)

Table 1: Experiment I - 3 h exposure - with and without metabolic activation

 

	- S9		+ S9
Concentration [µg/mL]	Relative Survival [%]	Mutant Frequency [per 10^6viable cells]	Relative Survival [%]	Mutant Frequency [per 10^6viable cells]
0 (Acetone)	100	210.32	100	225.19
31.25	100	255.07	124.7	174
62.5	103.6	244.61	122.1	210.76
125	104.3	278.49	105.8	250.95
250	102.9	215.07	114.4	205.16
NQO, 0.05	80.1	817.25	-	-
NQO, 0.1	95.2	600.74	-	-
B[a]P, 2	-	-	80.1	990.63
B[a]P, 3	-	-	64	1463.52

NQO: 4-nitroquinoline-N-oxide

B[a]P: Benzo[a]pyrene

Table 2: Experiment II - 3 h exposure – with and without metabolic activation

	- S9		+ S9
Concentration [µg/mL]	Relative Survival [%]	Mutant Frequency [per 10^6viable cells]	Relative Survival [%]	Mutant Frequency [per 10^6viable cells]
0 (Acetone)	100	200.06	100	173.79
31.25	101.9	148.05	101.5	174.39
62.5	91	194.93	303.7	237.18
125	100.7	168.76	107.5	174.80
250	95.8	218.93	101.5	172.87
NQO, 0.05	66.4	509.76	-	-
NQO, 0.1	88.4	689.34	-	-
B[a]P, 2	-	-	85	669.84
B[a]P, 3	-	-	67.1	1227.35

NQO: 4-nitroquinoline-N-oxide

B[a]P: Benzo[a]pyrene

Conclusions:

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

Reference 3

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:

1992-08-05, 1992-09-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (2-aminoanthracene was the only positive control compound used for reactions performed in the presence of metabolic activation).

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(2-aminoanthracene was used as only positive control with metabolic activation.)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UKEMS

Deviations:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA102

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

1.6, 8, 40, 200, and 1000 µg/plate (experiment 1) and 62.5, 125, 250, 500, and 1000 µg/plate (experiment 2)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

2-nitrofluorene

Remarks:

strain TA98

Migrated to IUCLID6: 50.0 µg/plate in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

sodium azide

Remarks:

strains TA100 and TA1535

Migrated to IUCLID6: 2.0 µg/plate in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

9-aminoacridine

Remarks:

strain TA1537

Migrated to IUCLID6: 50.0 µg/plate in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

other: Glutaraldehyde (25.0 µg/plate in water)

Remarks:

strain TA102

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminoanthracene 5.0 µg/plate in DMSO

Remarks:

at least applied in 1 strain

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, experiment 1) and preincubation (with metabolic activation, experiment 2)

DURATION
- Preincubation period: 1 hours at 37 °C in nutrient broth (experiment 2)
- Exposure duration: 72 h at 37 °C (for both: in agar (plate incorporation) and preincubation test)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: The background lawn was inspected for signs of toxicity.

Evaluation criteria:

The assay was considered valid if the following criteria were met:
i) the mean negative control counts fell within the normal range
ii) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation
iii) no more than 5% of the plates were lost through contamination or some other unforeseen event.
A test compound was considered to be mutagenic if:
i) the assay was valid
ii) Dunnett's test gave a significant response (p <0.01), and the data set showed a significant dose-correlation
iii) the positive responses described in (ii) were reproducible

Statistics:

For evaluation of test chemical and positive control data, the m-statistic was first calculated to check that the data were Poisson-distributed. Then, Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was then checked by linear regression analysis.

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

presumably as the result of the increased exposure of the bacteria to the test agent due to the pre-incubation step.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Yes, at 1000 µg/plate (experiment 1) and at 500 and 1000 µg/plate (experiment 2)

RANGE-FINDING/SCREENING STUDIES: Strain TA100 at 8, 40, 200, 1000, and 5000 µg/plate. No evidence of toxicity. Heavy precipitation of test agent was observed at concentrations of both 1000 and 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: Comparable

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: (experiment1)

Table 1: Revertants in S. typhimurium treated with the test substance, with and without metabolic activation (experiment 1):

Dose (µg/plate)	TA98		TA100		TA1535		TA1537		TA 102
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	22.2±5.7	17.2±6.1	115.2±6.4	124±3.2	21.8±2.9	26.6±4.3	17.8±2.1	13±3.5	232.6±21.1	305.4±39.5
1.6	27.7±5.1	24±7	105.7±6.7	119±29.5	26±1	29±1	15±2.6	10.3±3.1	298±50.7	362.3±7.8
8	29.7±5.9	21.3±7.4	95.3±9.5	113.7±9.1	23.7±10.4	18.7±7.6	14.3±4.6	9±2.6	265.3±11.7	317.3±33.6
40	25.3±6.7	21±6.1	113±2	124±23.8	17.7±2.5	23.7±1.2	16±5.3	11.7±3.5	267.7±39.7	333±28.6
200	29.0±2.6	22.7±8.4	108.7±11.9	99.3±24	21.7±1.2	28±4.4	15±1	11.3±3.5	283.3±26.5	316.7±15.3
1000	21.0±3.0	16.7±4.7	123.3±9.2	91±11	21.7±3.8	24±2.6	8.7±3.5	9.7±4	276±17.4	292±52.3
2-NF	1645.3±111.9	-	-	-	-	-	-	-	-	±
NaN3	-	-	810.3 ±57.4	-	463±57.7	-	-	-	-	-
AAC	-	-	-	-	-	-	425.7±9.1	-	-	-
AAN	-	1367.3±147	-	1800.3±249.7	-	195.7±4	-	-	-	-
GLU	-	-	-	-	-	-	-	-	477.7±45.8	-

(Mean ± SD)

  

2-NF: 2-nitrofluorene;

NaN3: sodium azide

GLU: glutaraldehyde

AAC: 9-aminoacridine

AAN: aminoanthracene

Table 2: Revertants in S. typhimurium, treated with the test substance, with and without metabolic activation (experiment 2):

Dose (µg/plate)

TA98

TA100

TA1535

TA1537

TA 102

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

29.4±5.0

28±4.5

111.6±12.7

103.6±10.8

22.2±2.8

21±1.6

15.4±3.8

13±2.2

319.2±48.9

357±19.4

62.5

34.7±1.2

21±1.7

109±13.5

83.7±5.1

21.3±1.2

19.7±3.2

19.3±2.5

18.3±0.6

271±43.4

316±16.9

125

36.7±7

28.7±6.1

113±10.5

87.3±10

16.3±3.5

11.3±4

16±0

13.7±2.1

222.3±11.9

311.3±13.4

250

36.3±5.5

34.3±1.2

102.7±3.1

90±28.3

21.3±5.7

22.3±3.1

17±2

14.3±3.1

237.3±27.6

351.3±11.4

500

19.3±9

24±4.6

90.3±1.5

70.7±2.1

15±5

21.3±3.8

15.3±2.1

16.3±4.5

292.3±29.8

162.7±11

1000

25.3±11.0

18.7±2.5

101±13.9

75.3±3.8

16.3±2.5

21±2.4

14.3±6.1

10.3±1.2

232.7±17.7

190.7±11

2-NF

1644±509.6

-

-

-

-

-

-

-

-

-

NaN3

-

-

773.3 ±75.1

-

479±49.4

-

-

-

-

-

AAC

-

-

-

-

-

-

643.3±227.7

-

-

-

AAN

-

2213.7±125.1

-

1451.7±38.1

-

-

-

-

-

-

GLU

-

-

-

-

-

-

-

-

715.3±89.4

-

(Mean ± SD)

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies

In a bacterial reverse mutation assay, conducted according to OECD Guideline 471, the test substance was evaluated for its mutagenic potential based on the ability to induce point mutations in selected loci of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102 (Hazleton 380/201, 1992). Dose levels from 1.6 up to 1000 µg/plate (experiment 1, plate incorporation) and 62.5 up to 1000 µg/plate (experiment 2, plate incorporation and preincubation) were investigated. Metabolic activation consisted of a cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254. Acetone was used as vehicle and solvent control. Appropriate positive controls were also evaluated, whereby 2-aminoanthracene was used as the only positive control with metabolic activation in at least one strain. Precipitation was observed at 1000 µg/plate (experiment 1) and at 500 and 1000 µg/plate (experiment 2). No effects of cytotoxicity were seen in experiment 1 but in experiment 2, presumably as the result of the increased exposure of the bacteria to the test agent due to the pre-incubation step. No dose-related biologically relevant increase or doubling in the number of his-revertants was observed in the plate incorporation test and in the pre-incubation in any of the tested strains with or without metabolic activation. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium mutation assay under the experimental conditions chosen. The negative and positive controls yielded the expected results.

A second bacterial reverse mutation assay was performed similar to OECD guideline 471, with TA98, TA100 and TA1537 at doses of 313 - 5000 µg/0.1 mL. The assay was performed in a plate incorporation test with and without metabolic activation and appropriate negative and positive controls were included. The assay yielded again no effects of genotoxicity (CIBA 894103, 1989).

Another bacterial reverse mutation assay was performed similar to OECD guideline 471, with S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100; E. coli WP2 uvr A at doses of 3.3 - 10000 µg/plate. The assay was performed in a plate incorporation test with and without metabolic activation and appropriate negative and positive controls were included. The assay yielded again no effects of genotoxicity (SRI intern.LSU-69 09, 1979).

Forward gene mutation induced by the test substance at the tk-locus was investigated in mouse lymphoma L5178Y cells according to OECD-guideline 476 at concentrations of 31.25, 62.5, 125, and 250 µg/mL in the presence and absence of a mammalian metabolic activation (cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254). Acetone was used as vehicle and solvent control and appropriate positive controls were also included in the assay. Precipitation was observed at 125 and 250 µg/mL. No cytotoxic effects were seen and there was no evidence of induced mutant colonies over background. The negative and positive controls induced the expected response (Hazleton 380/202, 1993).

In a mammalian cytogenetics assay (chromosome aberration), performed similar to OECD-guideline 473, CHO cell cultures were exposed to the test substance at concentrations of 29.4, 42, and 60 µg/mL (experiment I) and 60, 92.3, and 142 µg/mL (experiment II) with or without metabolic activation (cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254).

Acetone was used as vehicle control and cyclophosphamide as well as 4-nitroquinoline-N-oxide served as positive controls.

Precipitation at 39 µg/mL and above and no cytotoxicity to the CHO cells was observed. No chromosomal changes were evident in this assay. The negative and positive controls yielded the expected results (Hazleton 380/203, 1993).

In vivo studies

In a mammalian cytogenetics assay in vivo (chromosomal aberration), performed similar to OECD-guideline 475, rats (5 animals in the experimental groups and positive control group, 3 animals in the negative control group) were singly treated with test substance (intraperitoneal) at concentrations of 50, 500, and 5000 mg/kg bw (USFDA71-268, 1973). Two hours prior to killing, each animal was given 4 mg/kg of colcemid intraperitoneal in order to arrest the bone marrow cells in C-mitosis. Bone marrow cells were removed of one femur, prepared, fixed on a slide and evaluated.

Physiological saline was used as vehicle control and triethylenemelamine (0.3 mg/kg) served as positive control, both yielded the expected results.

No chromosomal changes were evident in this assay. In the same study, a second trial was perfomed. Differing from the procedure described above, in the second trial animals were treated on 5 consecutive days. Again, no chromosomal abberations were evident.

In a dominant lethal assay, performed similar to OECD-guideline 478, 10 male rats were treated with the test substance by gavage at concentrations of 50, 500 and 5000 mg/kg bw, singly or on 5 consecutive days (USFDA71 -268, 1973). Saline was used as vehicle control and triethylenmelamine (0.3 mg/kg bw, applied by intraperitoneal injection) as positive control. Both yielded the expected results. Dominant lethal effects were not observed.

Short description of key information:
No mutagenicity was observed in vitro, in GLP compliant studies with the test substance in a reverse mutation assay in bacteria (OECD 471, Hazleton 380/201), in a gene mutation assay in mouse lymphoma cells (OECD 476, Hazleton 380/202) and in a cytogenicity assay with CHO cells (OECD 473, 1993) nor in vivo (chromosomal aberration test and dominant lethal assay, USFDA 71-268).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. On the basis of the available data, the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.