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Genetic toxicity in vitro

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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:
15 April 2010 - 26 april 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium: Histidine geneE. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1:Preliminary test (with and without S9) TA100 and WP2uvrA: 3; 10; 33; 100; 333; 1,000; 3,330 and 5,000 µg/plate.Main study: TA1535, TA1537 and TA98Without and with S9-mix: 100; 333; 1,000; 3,330 and 5,000 µg/plate.Experiment 2:Without and with S9-mix: 100; 333; 1,000; 3,330 and 5,000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The test substance was soluble in DMSO. DMSO is accepted and approved by authorities and international guidelines.
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 Migrated to IUCLID6: (5 µg/plate in saline for TA 1535)
Positive control substance:
9-aminoacridine
Remarks:
without S9 Migrated to IUCLID6: (60 µg/plate in water for TA1537)
Positive control substance:
2-nitrofluorene
Remarks:
without S9 Migrated to IUCLID6: (10 µg/platein DMSO for TA98)
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: (6650 µg/platein DMSO TA100)
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 Migrated to IUCLID6: (10 µg/plate in DMSO for WP2uvrA)
Positive control substance:
other: 2-aminoanthracene inDMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)DURATION:- Exposure duration: 48 hoursNUMBER OF REPLICATIONS:- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.NUMBER OF CELLS EVALUATED: 10E8 per plateDETERMINATION OF CYTOTOXICITY- Method: Reduction of the bacterial background lawn, increase in the size of the microcolonies and reduction of the revertant colonies.OTHER EXAMINATIONS:- The presence of precipitation of the test substance on the plates was determined.
Evaluation criteria:
A test substance was considered negative (not mutagenic) in the test if:- The total number of revertants in tester strain TA100 was not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA 1535, TA1537, TA98 or WP2uvrA was not greater than three (3) times the concurrent control.- The negative response should have been reproducible in at least one independently repeated experiment.A test substance was considered positive if:- A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies was observed in the test substance group.- When a positive response was observed in one of the tester strains, the positive response should have been reproducible in at least one independently repeated experiment.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: No precipitation was observed up to and including the top dose of 5,000 µg/plate.RANGE-FINDING/SCREENING STUDIES:- No toxicity or mutagenicity was observed up to and including the top dose of 5,000 µg/plate.COMPARISON WITH HISTORICAL CONTROL DATA:- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.ADDITIONAL INFORMATION ON CYTOTOXICITY:- No toxicity or mutagenicity was observed up to and including the top dose of 5,000 µg/plate.
Conclusions:
Under the test conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method B.13/14.

Tester strains TA98, TA100, TA1535, TA1537 of Salmonella typhimurium strain and WP2uvrA of Escherichia coli in the presence and absence of S9 were exposed to the test substance. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to established the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluated and confirm the mutagenic potential of the test substance.

DMSO was selected as the solvent based on its solubility of the test substance and compatibility with the target cells. In the initial toxicity-mutation assay, the dose levels tested were 3; 10; 33; 100; 333; 1,000; 3,330 and 5,000 µg/plate. In the main study, the dose levels tested were 100; 333; 1,000; 3,330 and 5,000 µg/plate.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the test conditions, the test substance was found to be non-mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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:
From 21 March, 2013 to 20 August, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: UKEMS Guidelines
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine (guanine) phosphoribosyltransferase (Hprt) locus of mouse lymphoma L5178Y cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 media containing L-glutamine and HEPES- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S-9
Test concentrations with justification for top dose:
Experiment 1: 0, 100, 200, 300, 400 and 500 µg/mL in the absence of S-9; 0, 200, 400, 600, 750, 900, 1050 and 1,200 µg/mL in the presence of S-9Experiment 2: 0, 100, 200, 300, 400, 450 and 500 µg/mL in the absence of S-9; 0, 400, 600, 800, 950, 1050, 1100, 1150, 1200, 1300 and 1,400 µg/mL in the presence of S-9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: Test substance was miscible with analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 500 mg/mL. The solubility limit in culture medium was in excess of 5,000 μg/mL as indicated by a lack of any visible precipitation 3 h after test substance addition.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Exposure duration: 3 h- Expression time (cells in growth medium): 7 dSELECTION AGENT (mutation assays): 6-TGNUMBER OF REPLICATIONS: 4NUMBER OF CELLS EVALUATED: 20000 cells/well.DETERMINATION OF CYTOTOXICITY- Method: Plating efficiency (PE), Percentage relative survival (% RS), Adjusted % RS, Mutant frequency
Evaluation criteria:
For valid data, the test substance was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if: 1. The mutant frequency 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. The effects described above were reproducible.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:
All calculations were performed by computer using validated software. Statistical significance of mutant frequencies was carried out according to the UKEMS Guidelines (Robinson et al., 1990).
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
Range-finding/screening studies: The highest concentrations to give >10% relative survival (RS) were 625 μg/mL in the absence of S-9 and 1,250 μg/mL in the presence of S-9, which gave 10% and 29% RS, respectively. No marked changes in pH were observed in the range-finder experiment at the highest concentration tested (5,000 μg/mL) as compared to the concurrent vehicle controls (individual data not reported). Decreases in osmolality of more than 50 mOsm/kg (compared to the concurrent vehicle control value) were observed in the absence and presence of S-9 in the range-finder experiment at the highest concentration tested (5,000 μg/mL), but not at concentrations of 2500 μg/mL and below. As a precaution, further osmolality measurements were taken in Experiment 1 in the presence of S-9 only. Further measurements were not considered necessary in the absence of S-9 as the highest concentration tested was limited to 1000 μg/mL (individual data not reported).
Remarks on result:
other: strain/cell type: hprt locus
Remarks:
Migrated from field 'Test system'.

Toxicity:

In Experiment 1 twelve concentrations, ranging from 100 to 1,000 μg/mL in the absence of S-9 and from 200 to 2,500 μg/mL in the presence of S-9, were tested. Upon addition of the test substance to the cultures, evidence of precipitation was observed at the highest four concentrations in the absence of S-9 (650 to 1,000 μg/mL) and at the highest nine concentrations in the presence of S-9 (750 to 2,500 μg/mL) but no precipitate was observed following the 3 h treatment incubation period. Seven days after treatment, the highest seven concentrations in the absence of S-9 (550 to 1,000 μg/mL) and the highest five concentrations in the presence of S-9 (1,350 to 2,500 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentration analysed in the absence of S-9 was 500 μg/mL which yielded 10% RS. In the presence of S-9, no concentration gave 10-20% RS. Cultures treated at 1,050 and 1,200 μg/mL gave 21% and 8% RS, respectively, therefore both concentrations were analysed for viability and 6TG resistance. No marked changes in osmolality were observed at the highest concentration tested in the presence of S-9 in Experiment 1 (2,500 μg/mL), compared to the concurrent vehicle controls (individual data not reported). No further measurements of osmolality were made. In Experiment 2 ten concentrations, ranging from 50 to 750 μg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 200 to 1,400 μg/mL, were tested in the presence of S-9. Upon addition of the test substance to the cultures and following the 3 h treatment incubation period, no precipitate was observed at any concentration tested in the absence or presence of S-9. Seven days after treatment, the lowest concentration in the absence and presence of S-9 (50 and 200 μg/mL, respectively) were not selected as there were sufficient non-toxic concentrations and the highest three concentrations in the absence of S-9 (550 to 750 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 500 μg/mL in the absence of S-9 and 1400 μg/mL in the presence of S-9, which gave 13% and 10% RS, respectively.

Summary of results:

Experiment 1 (3 h treatment in the absence and presence of S-9):

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RS

MF§

 

%RS

MF§

0

 

100

2.54

 

0

 

100

2.18

 

100

 

77

3.00

NS

200

 

89

2.93

NS

200

 

54

3.27

NS

400

 

67

4.57

NS

300

 

44

4.23

NS

600

 

46

3.31

NS

400

 

29

5.65

NS

750

P

39

4.90

NS

500

 

10

3.03

NS

900

P

27

4.73

NS

 

 

 

 

 

1050

P

21

5.50

NS

 

 

 

 

 

1200

P,$$

8

(6.08)

 

Linear trend

NS

Linear trend

**

NQO

 

 

 

 

B[a]P

 

 

 

 

0.15

 

65

40.87

 

2

 

79

34.11

 

0.20

 

52

54.12

 

3

 

24

71.22

 

Experiment 2 (3 h treatment in the absence and presence of S-9):

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RS

MF§

 

%RS

MF§

0

 

100

4.65

 

0

 

100

3.80

 

100

 

106

2.75

NS

400

 

78

3.12

NS

200

 

61

4.57

NS

600

 

58

4.98

NS

300

 

38

3.67

NS

800

 

34

3.85

NS

400

 

22

6.70

NS

950

 

31

4.38

NS

450

 

19

3.05

NS

1050

 

22

2.81

NS

500

 

13

2.86

NS

1100

 

20

3.92

NS

 

 

 

 

 

1150

 

21

3.59

NS

 

 

 

 

 

1200

 

15

1.73

NS

 

 

 

 

 

1300

 

13

1.48

NS

 

 

 

 

 

1400

 

10

2.80

NS

Linear trend

NS

Linear trend

*

NQO

 

 

 

 

B[a]P

 

 

 

 

0.15

 

76

23.67

 

2

 

67

30.43

 

0.20

 

86

65.53

 

3

 

44

41.55

 

§-6‑TG resistant mutants/106viable cells 7 days after treatment

%RS-Percent relative survival adjusted by post treatment cell counts

$$- Data in parentheses indicates marked heterogeneity observed but included in analysis for comparative purposes

NS-Not significant

*, **, ***- Test for linear trend: χ2(one-sided), significant at 5%, 1% and 0.1% level respectively

P-Precipitation observed at the time of treatment

Conclusions:
Interpretation of results (migrated information):negativeUnder the test conditions, the test substance did not induce increases in mutant frequency at the hprt locus of mouse lymphoma L5178Y cells when tested up to highly toxic concentrations in the absence and presence of a metabolic activation system (S-9).
Executive summary:

An in vitro mouse lymphoma assay was conducted to evaluate the forward mutation induction potential at the HPRT locus in L5178Y mouse lymphoma cells according to OECD Guideline 476.

Based on the findings of the cytotoxic range-finder experiment, two independent experiments were conducted. A 3 h treatment incubation period was used for all experiments. The test substance was dissolved in DMSO. In the absence of S9, the maximum concentrations analysed were 500 µg/mL in Experiments 1 and 2. In the presence of S9, the maximum concentrations analysed were 1,200 (Experiment 1) and 1,400 (Experiment 2) µg/mL. In Experiments 1 and 2, no significant increases in mutant frequency (MF) were observed at any concentration analysed in the absence and presence of S9. Statistically significant linear trends were observed in the presence of S9 in Experiments 1 and 2 but, in the absence of any statistically significant increases in MF in both experiments, these observations were considered not biologically relevant. The highest concentration analysed in the presence of S9 in Experiment 1 (1,200 µg/mL) gave 8% relative survival but demonstrated significant heterogeneity between replicates. In Experiment 2, higher concentrations (up to a maximum of 1,400 µg/mL) were tested. All concentrations analysed in Experiment 2 gave acceptable levels of heterogeneity, therefore the data at 1,200 µg/mL in Experiment 1 was included in the report for comparative purposes.

Under the test conditions, the test substance did not induce increases in mutant frequency at the HPRT locus of mouse lymphoma L5178Y cells when tested up to highly toxic concentrations in the absence and presence of a metabolic activation system (S9) (Lloyd, 2013)

Endpoint:
in vitro cytogenicity / micronucleus study
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 December 2005 - 01 February 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: CrI:CD-1 (ICR)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River (UK) Limited, Margate, Kent- Age at study initiation: 5-8 wk- Weight at study initiation: 21-29 g- Assigned to test groups randomly: Yes- Housing: Seven animals/cage housed in solid-floor polypropylene cages with wood-flake bedding- Diet (e.g. ad libitum): Food (Certified Rat and Mouse Diet Code 5LF2, IPS Ltd., London, UK)- Water (e.g. ad libitum): Drinking water, ad libitum- Acclimation period: 7 d, ad libitumENVIRONMENTAL CONDITIONS- Temperature (°C): 19-25 °C- Humidity (%): 30-70 %- Air changes (per h): 15/h- Photoperiod (h dark / h light): 12 h dark / 12 h light
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Arachis oil- Concentration of test material in vehicle: 5, 25, 50 and 100 mg/mL- Amount of vehicle: 10 mL/kg- Lot/batch no. (if required): SN365
Duration of treatment / exposure:
24 or 48 hours
Frequency of treatment:
Single administration
Remarks:
Doses / Concentrations:250, 500 and 1,000 mg/kg bwBasis:nominal conc.
No. of animals per sex per dose:
Test and vehicle (control) group: Seven male micePositive control: Five male mice
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide- Route of administration: Oral- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
Bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Doses were selected based on the results of range-finding toxicity test in which 1,000 mg/kg was observed to be the maximum tolerated dose.DETAILS OF SLIDE PREPARATION: Immediately following termination (i.e. 24 or 48 h following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Griinwald/Giemsa, allowed to air-dry and cover-slipped using mounting medium.METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. Incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal; number of nonnochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes and NCE/PCE were recorded.
Evaluation criteria:
A positive mutagenic response would be demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48 hours kill times when compared to their corresponding control group.If these criteria were not fulfilled, then the test material was considered to be non-genotoxic under the conditions of the test.A positive response for bone marrow toxicity would be demonstrated when the dose group mean polychromatic to nonnochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by theUKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a (x + 1)^1/2 transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 2,000 mg/kg in range-finding test
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY- Dose range: 1,000 and 2,000 mg/kg- Mortality: 2/2 animals died at 2,000 mg/kg, no mortality at 1,000 mg/kg- Clinical signs of toxicity in test animals: Hunched posture, ptosis, ataxia, lethargy, pilo-erection and decreased respiratory rate.RESULTS OF DEFINITIVE STUDY- Induction of micronuclei (for Micronucleus assay): Increases in the frequency of micronucleated PCEs was statistically non-significant.- Ratio of PCE/NCE (for Micronucleus assay): Decreases in the PCE/NCE ratio in the 24 or 48 hours test material groups was statistically non-significant.

Table 1: Micronucleus Test - Summary of Group Mean Data

Treatment Group Number of PCE with Micronuclei per 2000 PCE PCE/NCE Ratio
Group Mean SD Group Mean SD
Vehicle Control (Arachis oil)
10 mL/kg
48-h Sampling Time
0.6 0.8 0.88 0.19
Vehicle Control (Arachis oil)
10 mL/kg
24-h Sampling Time
1 1 0.72 0.25
Positive Control (Cyclophosphamide)
50 mg/kg
24-h Sampling Time
49.0** 19.2 1.26 0.31
Amine Synergist
1000 mg/kg
48-h Sampling Time
0.6 1.1 0.8 0.32
Amine Synergist
1000 mg/kg
24-h Sampling Time
1.1 0.9 0.95 0.24
Amine Synergist
500 mg/kg
24-h Sampling Time
1.3 1.5 0.85 0.28
Amine Synergist
250 mg/kg
24-h Sampling Time
0.3 0.5 0.69 0.19

PCE = Polychromatic erythrocytes

NCE = Normochromatic erythrocytes

SD = Standard deviation

** = P < 0.01

Conclusions:
Under the test conditions, the test substance was negative in the mouse micronucleus test.
Executive summary:

A study was conducted to assess the potential of the test substance to produce damage to chromosomes or aneuploidy when administered to mice according to OECD Guideline 474 and EU Method B.12.

The test substance (250, 500 and 1,000 mg/kg bw) was administered intraperitoneally to seven male mice. Animals were killed 24 or 48 h later, the bone marrow extracted and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls, respectively.

There were no premature deaths seen in any of the dose groups. Clinical signs (hunched posture and ptosis) were observed in animals dosed with the test substance at and above 500 mg/kg bw in both the 24 and 48 h test groups. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48 h test material dose groups when compared to their concurrent control groups. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred and exposure to the bone marrow achieved. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Under the test conditions, the test substance was negative in the mouse micronucleus test.

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

Additional information

In vitro

A study was conducted to determine the mutagenic potential of amine synergist according to OECD Guideline 471 and EU method B.13/14 (Ames test). Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2uvrA were exposed to the substance in the presence and absence of S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase - the confirmatory mutagenicity assay - was to evaluated and confirm the mutagenic potential of the test substance. DMSO was selected as the solvent. In the initial toxicity-mutation assay, the dose levels tested were 3, 10, 33, 100, 333, 1,000, 3,330 and 5,000 µg/plate. In the main study, the dose levels tested were 100, 333, 1,000, 3,330 and 5,000 µg/plate. The negative and strain-specific positive control values were within the laboratory historical control data ranges. The substance was therefore determined to be non-mutagenic in the Ames reverse mutation assay (Verspeek-Rip, 2010).

An in vitro mouse lymphoma assay was conducted to evaluate the forward mutation induction potential at the HPRT locus in L5178Y mouse lymphoma cells according to OECD Guideline 476. Based on the findings of the cytotoxic range-finder experiment, two independent experiments were conducted. A 3 h treatment incubation period was used for all experiments. The test substance was dissolved in DMSO. In the absence of S9, the maximum concentrations analysed were 500 µg/mL in Experiments 1 and 2. In the presence of S9, the maximum concentrations analysed were 1,200 (Experiment 1) and 1,400 (Experiment 2) µg/mL. In Experiments 1 and 2, no significant increases in mutant frequency (MF) were observed at any concentration analysed in the absence and presence of S9. Statistically significant linear trends were observed in the presence of S9 in Experiments 1 and 2 but, in the absence of any statistically significant increases in MF in both experiments, these observations were considered not biologically relevant. The highest concentration analysed in the presence of S9 in Experiment 1 (1,200 µg/mL) gave 8% relative survival but demonstrated significant heterogeneity between replicates. In Experiment 2, higher concentrations (up to a maximum of 1,400 µg/mL) were tested. All concentrations analysed in Experiment 2 gave acceptable levels of heterogeneity, therefore the data at 1,200 µg/mL in Experiment 1 was included in the report for comparative purposes. Under the test conditions, the test substance did not induce increases in mutant frequency at the HPRT locus of mouse lymphoma L5178Y cells when tested up to highly toxic concentrations in the absence and presence of a metabolic activation system (S9) (Lloyd, 2013).

An in vitro cytogenetic study (according to OECD 473 or 487) was not conducted since in vivo testing is available (mouse micronucleus study; OECD 474).

In vivo

A study was conducted to assess the potential of the test substance to produce damage to chromosomes or aneuploidy when administered to mice according to OECD Guideline 474 and EU Method B.12. Amine synergist (250, 500 and 1,000 mg/kg bw) was administered intraperitoneally to seven males. Animals were killed 24 or 48 h later, the bone marrow extracted and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice) to serve as vehicle and positive controls, respectively. There were no premature deaths seen in any of the dose groups. Clinical signs (hunched posture and ptosis) were observed in animals dosed at and above 500 mg/kg bw in both the 24 and 48 h test groups. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48 h test material dose groups when compared to their concurrent controls. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred and exposure to the bone marrow achieved. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes, hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. The test substance was negative in the micronucleus test (Durward and Flanders, 2006).

Justification for classification or non-classification

Amine synergist was negative in in vitro and in vivo genetic toxicity tests. Therefore no classification is required for this endpoint according to CLP (EC 1272/2008) criteria.