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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: C12 -14 betaines, Negative for S typhimurium strains TA97a, TA98, TA100, TA1535, TA102 +/- S-9 (OECD TG 471)

Mammalian chromosome aberration test: Negative for human peripheral blood lymphocytes +/- S-9 (OECD TG 473)

Mammalian cell gene mutation: Negative for mouse lymphoma L5178Y cells +/- S-9 (OECD TG 476)

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
His-operon
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA1535, TA102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and β-Naphtoflavone induced rat liver S9
Test concentrations with justification for top dose:
1. Study: 0.016, 0.05, 0.16, 0.50, 1.60 mg/plate (TA97a, TA100 ± S9; TA1535 - S9); 0.05, 0.16, 0.50, 1.60, 5.00 mg/plate (TA98, TA102 ± S9; TA1535 + S9); 2. Study: 0.005, 0.016, 0.05, 0.16, 0.50 mg/plate (TA97a, TA102 ± S9; TA100, TA1535 - S9); 0.016, 0.05, 0.16, 0.50, 1.60 mg/plate (TA98, TA102 ± S9; TA100, TA1535 + S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
aqua bidest
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR 191, 0.5 µg/plate
Remarks:
TA97a without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
aqua bidest
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylene-diamine, 0.5 µg/plate
Remarks:
TA98 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
aqua bidest
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Nitrofurantoine, 0.2 µg/plate
Remarks:
TA100 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
aqua bidest
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 without S9Migrated to IUCLID6: 0.25 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
aqua bidest
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene, 2 µg/plate
Remarks:
TA97a, TA98, TA100, TA1535 with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
aqua bidest
True negative controls:
no
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
TA102 without S9Migrated to IUCLID6: 100 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
aqua bidest
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Danthron, 30 µg/plate
Remarks:
TA102 with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Exposure duration: 48 hoursSELECTION AGENT (mutation assays): histidine prototrophyNUMBER OF REPLICATIONS: triplicatesDETERMINATION OF CYTOTOXICITY - Method: other: reduction of background lawn
Evaluation criteria:
The test item was interpreted as mutagenic if a concentration effect relationship occurred and the induction rate was equal to or greater than 2. Spontaneous revertants/plate had to be within historical ranges, and the induction rates of the positive controls had to be equal to or greater than 2 to consider the test valid.
Statistics:
Arithmetic mean values and standard deviations were calculated from colonies per plate of three replicates. For evaluation of the results the induction rate of the mean values was calculated by division of the number of revertant colonies of the test item by the number of revertant colonies of the corresponding control.
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA1535, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Lowest cytotoxic concentration without and with S9 (mg/plate): TA97a (0.5 and 1.6, resp.); TA98 (1.6 and 5.0, resp.); TA100 (1.6 without S9 only); TA102 (5.0 and 5.0, resp.); TA1535 (1.6 and 5.0, resp.)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Test concentrations were selected based on the results of a preliminary test (non-GLP)COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous revertants/plate (negative controls) had to be within the following ranges: TA97a ± S9: 150 - 450; TA98 ± S9: 15 - 50; TA100 ± S9: 60 - 200; TA102 ± S9: 300 - 600; TA1535 ± S9: 5 - 30

Study 1:

Maximum number of revertants (mean ± SD):

Strain

-S9

+S9

Control

Test group (mg/plate)

Induction rate

Control

Test group (mg/plate)

Induction rate

TA97a

251 ± 10.5

288 ± 1.1 (0.16)

1.1

282 ± 15.3

252 ± 14.5 (0.16)

0.9

TA98

35 ± 7.1

33 ± 7.5 (0.05)

1.0

38 ± 6.1

39 ± 12.0 (0.16)

1.0

TA100

93 ± 8.2

104 ± 12.2 (0.016)

1.1

73 ± 8.2

82 ± 9.5 (0.5)

1.1

TA102

429 ± 8.3

444 ± 36.7 (0.5)

1.0

519 ± 16.3

519 ± 11.5 (0.16)

1.0

TA1535

18 ± 4.0

28 ± 3.1 (0.05)

1.5

7 ± 2.5

14 ± 3.6 (0.05)

1.9

 

Study 2:

Maximum number of revertants (mean ± SD):

Strain

-S9

+S9

Control

Test group (mg/plate)

Induction rate

Control

Test group (mg/plate)

Induction rate

TA97a

281 ± 26.1

239 ± 12.2 (0.05)

0.8

298 ± 41.6

273 ± 21.1 (0.05)

0.9

TA98

22 ± 10.4

41 ± 5.5 (1.6)

1.4

33 ± 2.6

35 ± 1.5 (0.5)

1.1

TA100

127 ± 14.2

111 ± 13.1 (0.016)

0.9

91 ± 8.0

91 ± 7.1 (0.5)

1.0

TA102

349 ± 20.4

355 ± 41.7 (0.05)

1.0

451 ± 34.3

462 ± 19.1 (0.5)

1.0

TA1535

29 ± 2.5

32 ± 1.7 (0.005)

1.1

11 ± 2.1

10 ± 3.1 (0.016)

0.9

 

Conclusion:

The test substance was not mutagenic to bacteria under the conditions chosen.

Conclusions:
The test item was not mutagenic in this assay
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Both the source and target substances are UVCB alkyldimethyl betaines. The Source substance has an alkyl chain length distribution comprising mainly C12-14, whilst the target chain length distribution is C12-16. It is considered that the presence of a greater percentage of C16 chain lengths in the target will not affect the mutagenic potential of the substance in this assay compared to the C12-14 source.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: betaines, C12-14 (even numbered) -alkyldimethyl [EC 931-700-2]
Target: betaines, C12-16 (even numbered) -alkyldimethyl [EC 947-036-1, CAS n/a]. Composition information per section 1.2

3. ANALOGUE APPROACH JUSTIFICATION
The Cosmetic Ingredient Review Expert Panel reviewed the product use, formulation and safety data of eleven alkyl betaines, as used in cosmetics. (Final report issued 2014-04-04)
The Panel concluded that the common core chemical structure, similar functions and concentrations in cosmetics, and the predicted physicochemical properties enabled grouping these ingredients and
reading across the available toxicological data to support the safety assessment of each individual compound in the entire group.
Therefore it is assessed that it is acceptable to apply the results of the study on cetyl betaine to the C12-16 betaine.

4. DATA MATRIX
Source: betaines, C12-14 (even numbered) -alkyldimethyl [EC 931-700-2]: OECD TG 471, negative
Target: betaines, C12-16 (even numbered) -alkyldimethyl [EC 947-036-1, CAS n/a]: OECD TG 473, negative; OECD TG 476, negative
Reason / purpose for cross-reference:
read-across source
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA1535, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Lowest cytotoxic concentration without and with S9 (mg/plate): TA97a (0.5 and 1.6, resp.); TA98 (1.6 and 5.0, resp.); TA100 (1.6 without S9 only); TA102 (5.0 and 5.0, resp.); TA1535 (1.6 and 5.0, resp.)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 January - 09 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
S-9 prepared from male Sprague-Dawley rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
Range-Finder:
3+17h, -S-9: 18.14 to 5000 μg/mL
3+17h, +S-9: 18.14 to 5000 μg/mL
20+0h, -S-9: 18.14 to 5000 μg/mL

Main experiment:
3+17h, -S-9: 10.00 to 125.0 μg/mL
3+17h, +S-9: 10.00 to 125.0 μg/mL
20+0, -S-9: 10.00 to 80.00 μg/mL

A maximum concentration of 5000 μg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to the maximum recommended concentration, for test articles of this type, according to current regulatory test guidelines. Concentrations selected for the chromosome aberration experiment were based on the results of this cytotoxicity Range-Finder Experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test material is sufficiently soluble for water to be used as the solvent.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 or 20 hours (-S9); 3 hours (+S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine

STAIN (for cytogenetic assays): 4% (v/v) Giemsa in pH 6.8 Gurr’s buffer

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Lymphocytes were kept in fixative at 2-8ºC prior to slide preparation for a minimum
of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, two to three minutes) and re-suspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of 45% (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading and several drops of suspension were transferred on to clean microscope slides labelled with the appropriate study details. Slides were flamed, as necessary, to improve quality. Slides were dried on a hot plate (set to approximately 80-100°C) and stained in filtered 4% (v/v) Giemsa in pH 6.8 Gurr’s buffer for 5 minutes. The slides were rinsed, dried and mounted with coverslips using DPX.

NUMBER OF CELLS EVALUATED: A minimum of 300 metaphases per concentration (150 metaphases from each code) were analysed for chromosome aberrations where possible.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range of one or more concentrations was observed in both replicate cultures
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p≤0.05)
3. A concentration-related increase in the proportion of cells with structural aberrations (excluding gaps) was observed (positive trend test).

The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were to be dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations, effects occurring only at high or very toxic concentrations and the types and distribution of aberrations.
Statistics:
After completion of scoring and decoding of slides the numbers of aberrant cells in each culture were categorised as follows:
1. Cells with structural aberrations including gaps
2. Cells with structural aberrations excluding gaps
3. Polyploid or endoreduplicated cells.

The totals for category 2 in vehicle control cultures were compared with the current historical vehicle control (normal) ranges to determine whether the assay was acceptable or not (see Acceptance Criteria). The proportion of cells with structural chromosome aberrations excluding gaps were compared with the proportion in vehicle controls by using Fisher’s exact test (Richardson et al., 1989). In addition, a
Cochran-Armitage Trend Test was performed to aid determination of concentration response relationships. Probability values of p≤0.05 were accepted as significant. The proportions of cells in categories 1 and 3 were examined in relation to the historical vehicle control ranges.
The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989). Probability values of p≤0.05 were accepted as significant.
Species / strain:
lymphocytes: human
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
Executive summary:

The substance was tested in an in vitrochromosome aberration assay using duplicate human lymphocyte cultures prepared from the pooled blood of three female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in purified water. The highest concentrations analysed in the Chromosome Aberration Experiment were limited by post-treatment precipitation (for the 3+17 hour treatment in the absence of S-9) or by toxicity (for the 3+17 hour treatment in the presence of S-9 and the 20+0 hour treatment in the absence of S-9) and were determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for chromosome analysis were selected by evaluating the effect of the substance on mitotic index. Chromosome aberrations were analysed at three concentrations.

Appropriate negative (vehicle) control cultures were included in the test system in the Chromosome Aberration Experiment under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within (or very close to) the current historical vehicle control (normal) ranges. Mitomycin C (MMC) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of rat liver S-9 respectively. Cells receiving these were sampled 20 hours after the start of treatment. Both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

All acceptance criteria were considered met and the study was accepted as valid.

Treatment of cultures with the substance for 3+17 hours in the

absence and presence of S-9 and for 20+0 hours in the absence of S-9 resulted in frequencies of cells with structural chromosome aberrations that were generally similar to and not significantly different (at the p≤0.05 level), compared to the concurrent vehicle controls, at all concentrations analysed under each treatment condition. Small, sporadic increases in structural chromosome aberration frequencies (excluding gaps), which marginally exceeded the normal ranges, were observed in single cultures at the highest concentration following the 3+17 hour treatment in the absence of S-9 (90 μg/mL) and at the lowest and highest concentrations following the 20+0 hour treatment in the absence of S-9 (10 and 30 μg/mL). However, the increases were not observed in the replicate cultures at these concentrations and there were no statistically significant linear trends under both treatment conditions. These isolated

observations were therefore considered not biologically relevant.

Small, sporadic increases in the frequencies of endoreduplicated and/or polyploid cells, compared to the concurrent vehicle controls and normal ranges, were observed under each treatment condition. However, this assay is not specifically designed to assess polyploidy, the observations were not quantitative and the increases were small and not necessarily concentration-related, therefore were considered of little or no biological relevance.

It is concluded that the substance did not induce biologically relevant increases in the frequency of structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested up to precipitating concentrations for 3+17 hours in the absence of a rat liver metabolic activation system (S-9) and when tested up to toxic concentrations for 3+17 hours in the presence of S-9 and 20+0 hours in the absence of S-9 under the experimental conditions described.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 January - 08 March 2019
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
Hprt
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Range finder 1 (-/+ S-9), 3-h treatment: 156.3, 312.5, 625, 1250, 2500, 5000 μg/mL
Range finder 2 (-/+ S-9), 3-h treatment: 1.563, 3.125, 6.25, 12.5, 25, 50, 100, 200 μg/mL
Mutation experiment (-S9), 3-h treatment: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 μg/mL
Mutation experiment (+S9), 3-h treatment: 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 80 μg/mL
Concentrations selected for the Mutation experiment were based promarliy on the results of the second range-finder experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Based on preliminary solubility data which indicated that the test item was soluble in purified water at concentrations up to at least 50.38 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Purified water diluted 10-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 or 8 days

SELECTION AGENT (mutation assays): 6-thioguanine


NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 2x10^4

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The mutation frequency (MF) at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF + 2 standard deviations).

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require 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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no data
- Water solubility: no
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:

In the first cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 156.3 to 5000 μg/mL. Overall toxicity was observed at all concentrations tested in the absence and presence of S-9.

In the second cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9, ranging from 1.563 to 200 μg/mL. The highest concentration to give >10% relative survival (RS) was 25 μg/mL, which gave 51% and 80% RS in the absence and presence of S-9, respectively .

No marked changes in osmolality or pH were observed in the first Range-Finder at the highest concentration tested (5000 μg/mL), compared to the concurrent vehicle controls.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
-S9
NQO, 0.15 μg/mL mean MF = 26.44, range 0-5.96 (mutants per 10^6 viable cells)
NQO, 0.20 μg/mL mean MF = 29.61, range 2.52-56.71 (mutants per 10^6 viable cells)
+S9
B[a] P, 2 μg/mL mean = 23.91, range 4.74 to 43.08 (mutants per 10^6 viable cells)
B[a] P, 3 μg/mL mean = 23.60, range 1.79 to 45.40 (mutants per 10^6 viable cells)

- Negative (solvent/vehicle) historical control data:
-S9 mean = 2.98, range 0 to 5.96 (mutants per 10^6 viable cells)
+S9 mean = 2.82, range 0.45 to 5.20 (mutants per 10^6 viable cells)
Conclusions:
The test item is considered to be not mutagenic, with and without metabolic activation, in this test.
Executive summary:

The test item was assayed to assess any potential to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test item was formulated in purified water.

A 3-hour treatment incubation period was used for each experiment.

In an initial cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 156.3 to 5000 μg/mL (an acceptable maximum concentration for testing mixtures in in vitro genetic toxicology studies of this type, according to current regulatory test guidelines). Overall toxicity was observed at all concentrations tested in the absence and presence of S-9.

In the second cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9, ranging from 1.563 to 200 μg/mL. The highest concentration to give >10% relative survival (RS) was 25 μg/mL, which gave 51% and 80% RS in the absence and presence of S-9, respectively.

In the Mutation Experiment eleven concentrations, ranging from 5 to 60 μg/mL, were tested in the absence of S-9 and twelve concentrations, ranging from 5 to 80 μg/mL, were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 45 μg/mL in the absence of S-9 and 60 μg/mL in the presence of S-9, which gave 4% and 9% RS,

respectively. Extremely steep concentration-related toxicity was observed under both treatment conditions. In the absence of S-9, concentrations of 40 and 45 μg/mL gave 26% and 4% RS, respectively and in the presence of S-9, concentrations of 55 and 60 μg/mL gave 23% and 9% RS, respectively. Both concentrations were analysed for viability and 6TG resistance under both treatment conditions.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore, the study was accepted as valid.

When tested up to highly toxic concentrations in the absence of S-9, no statistically significant increases in MF were observed following treatment with Amphosol CDB special dried at any concentration analysed and there was no statistically significant linear trend.

When tested up to highly toxic concentrations in the presence of S-9, statistically significant increases in MF were observed at two intermediate concentrations of 25 μg/mL and 55 μg/mL, with a weak statistically significant linear trend. However, no such increases in MF were observed at intermediate concentrations between 30 and 50 μg/mL (giving 78% to 36% RS) or at the maximum concentration of 60 μg/mL, which gave 9% RS. The mean MF value at 25 μg/mL was 5.71 mutants per 106 viable cells (individual values 5.40 and 6.04) and at 55 μg/mL the mean MF value was 6.16 mutants per 106 viable cells (individual values 4.73 and 7.51). At the time of this experiment, the historical vehicle control range in the presence of S-9 (based on the last 20 experiments prior to this one) was 0.45 to 5.20 mutants per 106 viable cells, therefore both mean MF values were marginally outside the range but one of the replicate cultures at 55 μg/mL was within the range. Furthermore, although there was a weak but statistically significant linear trend (p≤0.05), there was no clear concentration-related mutagenic response over the concentration range analysed.

These results were therefore considered to be of doubtful biological relevance.

It is concluded that the test item showed evidence of inducing weak mutagenic activity at the hprt locus of L5178Y mouse lymphoma cells at 2 out of 10 concentrations tested in the presence of a rat liver metabolic activation system (S-9). However, as the increases in mutant frequency were limited to only two intermediate concentrations, were small in magnitude and were not clearly concentration-related, they were therefore considered of doubtful biological relevance.

In the same test system, the test item did not induce mutation when tested up to highly toxic concentrations for 3 hours in the absence of S-9 under the experimental conditions described.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The substance or its analogue were negative in several in vitro studies with and without metabolic acitivation using S-9, namely an OECD TG 471, OECD TG 473, OECD TG 476. Classification is not required.