Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- negative: Ames test with S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvr A (met. act.: with and without) (OECD TG 471; GLP); cytotoxicity: yes

- negative: Mammalian cell gene mutation assay with mouse lymphoma L5178Y cells (TK) (met. act.: with and without) (OECD TG 476; GLP); cytotoxicity: yes; read across from Quaternary ammonium compounds, Benzylbis(hydrogenated tallow alkyl)methyl, chlorides

- negative: In vitro mammalian chromosome aberration test with V79 Chinese Hamster cells (met. act.: with and without) (OECD TG 473; GLP); cytotoxicity: yes; read across from DODMAC

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his (Salmonella typhimurium strains), trp (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.316, 1.0, 3.16, 10.0, 31.6 and 100 μg
Pronounced cytotoxicity was noted starting at a concentration of 100 μg/plate in the preliminary cytotoxicity tests.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test item was not soluble in highly purified water or dimethylsulfoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: 1st experiment: in agar (plate incorporation); 2nd experiment: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: background lawn



Evaluation criteria:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for the Salmonella typhimurium test strains TA98, TA100, TA1535, TA1537 and Escherichia coli test strain WP2 uvrA in both independent experiments.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman’s rank correlation coefficient) is observed;
Biological relevance of the results should be considered first.
Positive results have to be reproducible and the histidine or tryptophan independence of the revertants has to be confirmed by streaking random samples on histidine or tryptophan-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Precipitation: not reported

HISTORICAL CONTROL DATA
- see attachment

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants) were noted at the top concentration of 100 μg/plate, in all Salmonella typhimurium strains and in the Escherichia coli strain WP2 uvrA [pKM101].

Plate incorporation, without metabolic activation

 

 

TA98

TA100

TA1535

TA1537

E. coli WP2

0.316

mean

SD

27.3

1.2

117.0

2.6

15.3

1.5

6.3

1.2

30.0

0.0

1.0

mean

SD

36.3

0.6

118.7

4.6

17.0

1.0

5.7

2.1

29.3

2.9

3.16

mean

SD

36.3

1.5

127.0

13.1

17.3

1.5

7.7

0.6

37.7

8.7

10

mean

SD

39.0

6.1

128.3

7.6

16.7

1.5

5.7

1.2

45.3

6.4

31.6

mean

SD

29.0

15.5

106.3

2.1

18.0

1.7

9.0

1.0

53.7

6.8

100

mean

SD

12.0 #

1.0

66.7 #

0.6

8.7 #

1.2

2.0 #

0.0

14.3 #

0.6

Negative control

mean

SD

29.0

6.2

131.3

15.4

25.0

0.0

6.7

0.6

51.3

3.1

Positive control

mean

SD

143.3

1.5

990.3

4.9

149.3

0.6

97.3

9.5

205.3

4.9

Plate incorporation, with metabolic activation

0.316

mean

SD

33.3

1.2

143.3

17.0

17.3

2.1

5.3

2.3

42.7

2.1

1.0

mean

SD

26.7

5.5

131.0

15.6

20.7

7.4

7.0

1.7

45.7

6.4

3.16

mean

SD

33.0

4.6

128.0

22.3

17.7

0.6

7.0

1.7

47.0

4.6

10

mean

SD

25.7

2.5

131.0

2.6

17.7

0.6

7.3

1.2

41.3

4.0

31.6

mean

SD

31.0

7.8

129.3

3.2

16.3

0.6

7.0

0.0

39.0

14.4

100

mean

SD

12.7 #

1.2

68.0 #

3.6

8.3 #

0.6

2.0 #

0.0

14.3 #

1.5

Negative control

mean

SD

36.7

0.6

120.7

2.1

17.0

1.0

6.7

1.2

48.3

12.1

Positive control

mean

SD

145.7

4.0

980.0

30.8

152.3

10.4

85.0

17.3

184.7

35.9

Preincubation, without metabolic activation

0.316

mean

SD

30.7

4.7

152.7

7.6

19.0

1.0

6.3

0.6

45.0

1.7

1.0

mean

SD

26.0

9.5

164.3

18.5

22.0

2.6

7.0

1.0

53.3

5.7

3.16

mean

SD

30.7

9.3

160.7

22.9

21.7

1.5

7.3

1.2

52.7

2.1

10

mean

SD

41.7

6.4

167.3

12.4

24.7

4.0

7.3

0.6

36.0

10.4

31.6

mean

SD

31.0

1.7

108.7

5.9

24.3

3.1

6.3

0.6

43.0

1.0

100

mean

SD

14.7 #

0.6

51.0 #

1.0

7.7 #

0.6

2.0 #

0.0

15.7 #

0.6

Negative control

mean

SD

28.3

3.2

174.0

3.6

27.7

0.6

5.3

0.6

41.7

18.9

Positive control

mean

SD

146.0

3.6

845.3

4.0

193.0

1.0

86.3

2.3

143.0

28.2

Preincubation, with metabolic activation

0.316

mean

SD

30.7

3.5

139.3

24.6

25.0

4.6

6.3

2.1

50.3

1.5

1.0

mean

SD

26.3

4.7

138.7

28.5

23.7

4.6

6.3

2.1

54.3

4.2

3.16

mean

SD

29.7

5.5

111.7

1.2

20.0

0.0

6.3

1.2

57.7

1.2

10

mean

SD

29.0

4.6

122.0

4.6

22.0

3.6

4.7

0.6

54.0

7.0

31.6

mean

SD

23.3

3.1

109.7

2.1

21.0

2.0

8.0

1.7

44.7

 

0.6

100

mean

SD

13.7 #

1.5

59.3 #

8.3

7.3 #

0.6

2.0 #

0.0

15.3 #

1.2

Negative control

mean

SD

31.7

9.0

177.3

33.2

22.7

1.5

7.7

1.5

49.7

1.5

Positive control

mean

SD

137.3

5.7

830.3

16.2

153.0

13.0

80.3

5.5

159.7

3.1

Conclusions:
Di-C12-18 alkyldimethyl ammonium chloride was not mutagenic in this bacterial reverse mutation assay in the presence and absence of metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997) and EU method B.13/14 (2008), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvr A were exposed to Di-C12-18 alkyldimethyl ammonium chloride in ethanol in concentrations of 0 (control), 0316, 1.0, 3.16, 10.0, 31.6 and 100 µg/plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method (1st experiment) and pre-incubation method (2nd experiment; 20 min pre-incubation).

The test substance was tested up to cytotoxic concentrations. Pronounced cytotoxicity was noted at 100 μg/plate in in all strains.

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures were within the range of the historical data. Hence, all acceptance criteria are met.

No increase in revertant colony numbers as compared with control counts was observed for the test item in the Salmonella typhimurium and in the Escherichia coli test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

Under the conditions of the study, the test substance was negative for mutagenic potential.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
• they are manufactured from similar or identical precursors under similar conditions
• they share structural similarities with common functional groups: quaternary ammonium and saturated or unsaturated alkyl chains with comparable length (corresponding to scenario 2 of the read-across assessment framework)

The read-across hypothesis is based on structural similarity of target and source substances. Based on available experimental data, including key physicochemical properties and data from acute toxicity, irritation, sensitization (human) and genotoxicity studies, the read-across strategy is supported by a quite similar toxicological profile of all substances.

Therefore, read-across from the existing ecotoxicity, environmental fate and toxicity studies conducted with the source substances is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

A justification for read-across is attached to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See justification for read-across attached to IUCLID section 13.

3. ANALOGUE APPROACH JUSTIFICATION
See justification for read-across attached to IUCLID section 13.

4. DATA MATRIX
See justification for read-across attached to IUCLID section 13.
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across source
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
18 and 28 hours after treatment at 40 µg/ml without S9 mix
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative with and without S9


Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
• they are manufactured from similar or identical precursors under similar conditions
• they share structural similarities with common functional groups: quaternary ammonium and saturated or unsaturated alkyl chains with comparable length (corresponding to scenario 2 of the read-across assessment framework)

The read-across hypothesis is based on structural similarity of target and source substances. Based on available experimental data, including key physicochemical properties and data from acute toxicity, irritation, sensitization (human) and genotoxicity studies, the read-across strategy is supported by a quite similar toxicological profile of all substances.

Therefore, read-across from the existing ecotoxicity, environmental fate and toxicity studies conducted with the source substances is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

A justification for read-across is attached to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See justification for read-across attached to IUCLID section 13.

3. ANALOGUE APPROACH JUSTIFICATION
See justification for read-across attached to IUCLID section 13.

4. DATA MATRIX
See justification for read-across attached to IUCLID section 13.
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across source
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 mix ≥ 50 µg/mL at the 3-hour treatment; ≥ 10 µg/mL at the 24-hour treatment with S9 mix ≥ 100 µg/mL at the 3-hour treatment
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative with and without S9 mix

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A reverse gene mutation assay in bacteria according to OECD guideline 471 is available for the target substance Di-C12-18 alkyl-dimethyl ammonium chloride. A mammalian cell gene mutation assay, and an in vitro mammalian chromosome aberration test are available for the structurally closely related source substances Quaternary ammonium compounds, Benzylbis(hydrogenated tallow alkyl)methyl, chlorides and DODMAC, respectively. A justification for read-across is attached to IUCLID section 13.

 

in vitro gene mutation study in bacteria

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997) and EU method B.13/14 (2008), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvr A were exposed to Di-C12-18 alkyldimethyl ammonium chloride in ethanol in concentrations of 0 (control), 0316, 1.0, 3.16, 10.0, 31.6 and 100 µg/plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method (1st experiment) and pre-incubation method (2nd experiment; 20 min pre-incubation).

The test substance was tested up to cytotoxic concentrations. Pronounced cytotoxicity was noted at 100 μg/plate in in all strains.

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures were within the range of the historical data. Hence, all acceptance criteria are met.

No increase in revertant colony numbers as compared with control counts was observed for the test item in the Salmonella typhimurium and in the Escherichia coli test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

Under the conditions of the study, the test substance was negative for mutagenic potential.

 

The source substance DODMAC as well was not mutagenic in a reverse gene mutation assay in bacteria. The preliminary assay with or without metabolic activation showed that the test substance demonstrated a potent toxicity from 1000 to 2500 µg/plate in all Salmonella strains tested. Under these conditions, the dose of 1000 µg/plate was retained as the maximum dose tested for the mutagenicity assay in Salmonella strains. In Escherichia Coli WP2uvrA, the top dose was 2500 µg/plate.

No precipitate was observed in the petri plates when scoring the revertants at all dose-levels. Without metabolic activation, toxicity was observed at the dose level of 1000 µg /plate for TA 1537, TA 1538, TA 98 and TA 100 strains. In Escherichia Coli WP2uvrA, toxicity was noted at 2500 µg /plate. With metabolic activation, cytotoxicity was limited to the strain TA 1537 which exhibited toxicity at the highest dose-level of 1000 µg/plate.

No significant increase in the mean number of revertants was noted in the five Salmonella typhimurium strains and Escherichia coli tested in the presence of the test substance neither with nor without metabolic activation. It was concluded that DODMAC was not mutagen under the conditions of the study.

 

in vitro gene mutation study in mammalian cells

The potential for Quaternary ammonium compounds, Benzylbis(hydrogenated tallow alkyl)methyl, chlorides to induce mutations at the TK locus, was investigated in L5178Y mouse lymphoma cells. The test substance was tested in two independent experiments, both with and without metabolic activation. Approximately 0.5 x 106(3-hour treatment) or 0.15 x 106(24-hour treatment) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main experiments was based on the level of toxicity (decrease in Adj. RTG), according to the criteria specified in the international guidelines.

In the experiments without metabolic activation, the selected dose-levels were as follows:

 2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the first experiment (3-hour treatment),

1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL for the second experiment (24hour treatment).

Cytotoxicity was observed. Following the 3-hour treatment, a marked to severe toxicity was induced at dose-levels ≥ 37.5 µg/mL, as shown by a 61-100% decrease in Adj. RTG. Following the 24-hour treatment, a marked to severe toxicity was induced at doselevels ≥ 6.25 µg/mL, as shown by a 80-100% decrease in Adj. RTG.

No noteworthy increase in the mutation frequency was noted in comparison to the vehicle control following the 3-hour or the 24-hour treatments.

In the experiments with metabolic activation, the selected dose-levels were as follows:

4.69, 9.38, 18.75, 37.5, 75 and 150 µg/mL for the first experiment,

2.34, 4.69, 9.38, 18.75, 37.5 and 75 µg/mL for the second experiment.

 In the first experiment, a slight to strong precipitate was noted in the culture medium at the end of the 3-hour treatment at dose-levels ≥ 4.69 µg/mL.

Cytotoxicity was observed. In the first experiment, a severe toxicity was induced at dose-levels ≥ 75 µg/mL, as shown by a 88-100% decrease in Adj. RTG. In the second experiment, a moderate to severe toxicity was induced at dose-levels ≥ 37.5 µg/mL, as shown by a 49-100% decrease in Adj. RTG.

In either experiment, no noteworthy increase in the mutation frequency was noted in comparison to the vehicle control. 

 

in vitro chromosome aberration study in mammalian cells

In the Hoechst AG study (1989), DODMAC (90% active in isopropanol/water) was examined for its genotoxic activity in V79 Chinese Hamster cells. The induction of the chromosome aberrations after in vitro treatment was investigated in the presence and in the absence of S9 mix.

A preliminary cytotoxicity experiment was performed in order to select the appropriate dose-levels for the main experiment. The test substance produced a significant cytotoxic effect (reduction of plating efficiency) without metabolic activation from 50 µg/ml up to a concentration of 200 µg/ml which was the limit of solubility.

In the main experiment, two independent cell cultures with and without metabolic activation (S9 -mix) were used with the dose-levels of 4, 20 and 40 µg/ml in the absence of metabolic activation and 5, 25 and 50 µg/ml in the presence of metabolic activation.

The test substance did not induce increase in the number of metaphases with aberration at any preparation time and dose-level.

A cytotoxic effect was observed 18 and 28 hours after treatment at 40 µg/ml without metabolic activation. Marked increases in the rate of chromosome aberrations were observed with the positive controls indicating the sensitivity of the assay.

In conclusion, DODMAC does not induce chromosome mutations (aberrations) in V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system.

Justification for classification or non-classification

Based on the available data, the target substance Di-C12-18 alkyl-dimethyl ammonium chloride does not need to be classified and labelled according to the CLP Regulation (EC) No 1272/2008 with respect to mutagenicity.