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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro (Mutagenic effects - bacterial): NOAEL; OECD 471; Ames study; mutagenic; reliability = 2.
NOT CLASSIFIED

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
No reference to OECD guideline. Deviation from OECD guideline with the use of 4-o-Tolylazo-o-toluidine as positive control. Study conducted in 1975.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only 4 strains of bacteria were used. Strain TA1538 was used and Strain TA98 was not. Positive controls deviate from those recommended in guideline.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitol-stimulated rat liver homogenate
Test concentrations with justification for top dose:
1, 10, 50, 100, 250, 500, 750, 1000 micrograms/plate.
Vehicle / solvent:
no data
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-o-Tolylazo-o-toluidine (for TA1538 and metabolic activation) and N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
In the absence of metabolic activation, approximately 5E8 bacteria were added to 2 mL of top agar (0.6% Difco agar, 0.6% NaCl, 0.050 mM of L-histidine, 0.05 mM of biotin). After the test chemical was added to the top agar, the solution was mixed and poured onto the surface of a minimal agar plate with Vogel-Bonner E medium containing 1.5% agar and 2% glucose.

The metabolic activation system consisted of 0.15 mL of the 9000 x g supernatant of homogenized rat liver, MgCl2, KCl, glucose-6-phosphate, TPN, and a sodium phosphate buffer (pH 7.4). This mixture (0.25 mL) was added directly to the top agar immediately before it was poured over the minimal agar plate. The plates were incubated at 37 deg C for 40 hours. The number of histidine-positive (his+) revertant colonies on each plate then was counted. All tests were performed in duplicate.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION: 40 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF CELLS EVALUATED: 5E8
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified

In Vitro Salmonella Typhimurium Assay With m-PDA.

 

Chemical

Metabolic Activation

Amount of Compound Added per Plate

(µg)

Average Number of Histidine-Positive Revertants per Plate

TA100

TA1535

TA1537

TA1538

Negative Control

-

 

72

11

5

9

+

 

84

11

6

15

 

 

 

 

 

 

 

4-o-Tolylazo-o-toluidine

(Positive control for TA1538 and metabolic activation)

-

25

 

 

 

10

+

25

 

 

 

195

 

 

 

 

 

 

 

N-methyl-N’-nitro-N-nitrosoguanidine

-

2

 

600

 

 

 

 

 

 

 

 

 

m-phenylenediamine

-

1

65

9

4

4

 

-

10

64

8

4

2

 

-

50

78

9

5

9

 

-

100

80

10

3

12

 

-

250

 

 

 

9

 

-

500

83

8

2

17

 

-

750

 

 

 

 

 

-

1000

74

11

5

21

 

+

1

87

14

8

10

 

+

10

78

11

7

17

 

+

50

80

9

6

79

 

+

100

85

14

6

177

 

+

250

 

 

 

423

 

+

500

84

14

11

570

 

+

750

 

 

 

623

 

+

1000

79

10

8

971

 

Conclusions:
The test substance was mutagenic in strain TA 1538 with metabolic activation in the S. typhimurium assay, but negative when activation was not used. It was negative in all other strains, with or without activation.
Executive summary:

The test substance was mutagenic in strain TA 1538 with metabolic activation in the S. typhimurium assay, but negative when activation was not used. It was negative in all other strains, with or without activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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
Endpoint:
in vitro gene mutation study in mammalian cells
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In Vivo (Clastogenic effects - mammalian): NOAEL; OECD 474; GLP; In vivo mouse micronucleus study; no mammalian mutagenesis was observed at any exposure level; reliability = 2.
NOT CLASSIFIED

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 6, 1990 to January 9, 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment. Conclusion based on scoring 1000 PCEs/animal for MNPCEs versus 2000 PCEs/animal as per OECD 474.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
scored fewer PCEs/animal than recommended.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Crl:CD-1(ICR)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Canada Inc., Breeding Area 62, Montreal, Quebec, Canada.
- Age at study initiation: 50 days old
- Weight at study initiation: Males were 28.6 to 33.5 g, females were 21.3 to 26.1 g
- Assigned to test groups randomly: By computer-generated random numbers
- Housing: Individually housed in standard wire mesh cages
- Diet: Purina certified Rodent Chow #5002, Lots # Aug 07 901A and Aug 10 901B) ad libitum
- Water: Ad libitum
- Acclimation period: Quarantined and acclimated for 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): 50 ± 10
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark


Route of administration:
oral: gavage
Vehicle:
Deionized water
Frequency of treatment:
Twice, with doses administered approximately 24 hours apart.
Remarks:
Doses / Concentrations:
16, 33, 65 mg/kg/day
Basis: actual ingested
No. of animals per sex per dose:
5 males/females per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
Details of tissue and slide preparation:
Immediately following sacrifice at 24 or 48 hours after the administration of the second dose, the marrow from both femurs of each animals was collected. A Miniprep automatic blood smearing instrument was used to make the bone marrow smears. At least 2 slides/animal were prepared.
Evaluation criteria:
Representative slides from each animal were examined in a blind manner using incident light fluorescence microscopy. Only cells showing good morphology and staining were selected for scoring. Polychromatic erythrocytes (PCEs) were identified by their characteristic reddish staining. Normochromatic erythrocyte (NCEs) appeared dark green. One thousand PCEs/animal were scored for the presence of micronuclei, which are typically round, bright yellow-green fluorescent bodies. Cellular inclusions that were irregularly shaped or stained, or were not in the focal plane of the cell were considered artifacts. The unit of scoring was the micronucleated cell. PCEs containing more than one micronucleus were counted as a single micronucleated cell. The number of micronucleated NCEs seen in the optic fields scored to obtain 1000 PCEs was also recorded. The proportion of PCEs among 1000 erythrocytes was determined for each animal.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 25, 50, 65, 75 mg/kg/day, 2X ~24 hours apart.

- Clinical signs of toxicity in test animals: 75 mg/kg/day group, 3/3 males and 1/3 females died within 30 hours of second dosing.
65 mg/kg/day-lethargic and had labored breathing; these signs were observed in all animals and occurred within 2 hrs of dosing on both days. Mild clinical signs (ruffled fur and partially closed eyes) persisted for 2 days following the final exposure, however, all animals recovered within 4 days post-treatment. 50 and 25 mg/kg/day-face pawing, labored breathing, gasping, lethargy and half closed eyes.

- Dose range: 85, 100, 200 or 300 mg/kg-a second treatment at these levels was not conducted due to excessive mortality or the severity of the clinical signs observed 24 hrs after the initial treatment


RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: 65 mg/kg/day-within 1 hour of initial dose, mice exhibited lethargy, gasping, face pawing, spasms, tremors, or half-closed eyes. Lethargy, half-closed eyes and labored and/or rapid breathing were still evident in the majority of the animals, 3-4 hrs after initial observations. Clinical signs following the second dose were similar to those observed the preceding day. One male was found dead 24 hrs after the final treatment. 16 and 33 mg/kg/day-lethargy, half closed eyes and rapid breathing within 24 hrs of final dose. The majority of the 16 mg/kg/day treated group appeared to recover, and only slight clinical sign were still evident in the 33 mg/kg/day treated group.

Weight loss-there was a statistically significant weight loss in 33 and 65 mg/kg/day treated groups at the 24 hour sacrifice, and in the 65 mg/kg/day treated group at the 48 hr sacrifice.

There were no statistically significant increases in MN-PCEs among test substance treated mice at any dose level or sampling interval.

Table 1. Mouse Micronucleus Assay of m-PDA

 

m-PDA Treatment

(mg/kg/day)

Sampling Time

(hrs)

Sex

Animals Per Group

Change in Body Weight (g)

x ± S.E.

0

24

M

5

0.0 ± 0.2

 

 

F

5

-0.4 ± 0.5

16

24

M

4a

-0.9 ± 0.6

 

 

F

5

-0.7 ± 0.4

33

24

M

5

-3.3 ± 0.7**

 

 

F

5

-2.8 ± 0.2**

65

24

M

6

-5.3 ± 0.4**

 

 

F

6

-4.5 ± 0.3**

0

48

M

5

0.4 ± 0.4

 

 

F

5

0.8 ± 0.9

65

24

M

4a,b

-3.4 ± 0.9**

 

 

F

6

-3.3 ± 0.5**

CP, 20

24

M

5

0.2 ± 0.3

 

 

F

5

-0.6 ± 0.2

 

 

 

 

 

 

CP = cyclophosphamide

a animal misdosed and removed from study.

b animal died before scheduled sacrifice time.

** p < 0.01

 

 

Table 2. Data Summary. Mouse Micronucleus Assay of m-PDA.

 

m-PDA Treatement

(mg/kg/day)

Sampling Time

(hrs)

Mean % Micronucleated PCEs ± S.E.a

 

Mean PCE:NCE Ratio ± S.E.

N

Males

N

Females

 

Males

 

Females

0

24

5

0.16 ± 0.05

5

0.02 ± 0.02

 

1.16 ± 0.11

 

1.12 ± 0.03

16

24

4b

0.15 ± 0.09

5

0.16 ± 0.05

 

0.96 ± 0.05

 

1.13 ± 0.08

33

24

5

0.24 ± 0.07

5

0.10 ± 0.04

 

0.91 ± 0.07

 

1.14 ± 0.07

65

24

6

0.28 ± 0.07

6

0.13 ± 0.05

 

0.77 ± 0.10

 

1.13 ± 0.12

 

 

 

 

 

 

 

 

 

 

0

48

5

0.40 ± 0.13

5

0.28 ± 0.05

 

0.98 ± 0.12

 

1.34 ± 0.09

65

48

4b,c

0.50 ± 0.27

6

0.18 ± 0.03

 

0.58 ± 0.08*

 

0.87 ± 0.26

 

 

 

 

 

 

 

 

 

 

CP, 20

24

5

0.96 ± 0.09**

5

0.82 ± 0.09**

 

0.71 ± 0.03**

 

1.00 ± 0.10

 

 

 

 

 

 

 

 

 

 

 

CP = cyclophosphamide

a 1000 PCEs scored per animal.

b animal misdosed and removed from study.

c animal died before scheduled sacrifice time.

*   p < 0.05

** p < 0.01

 

Conclusions:
Mice exposed to the test substance did not exhibit statistically significant increases in micronucleated polychromatic erythrocytes at any dose level or sampling interval.
Executive summary:

No statistically significant increases in the frequency of micronucleated PCEs were observed in test substance-treated animals at any sampling time. A significant depression in the ratio of young, polychromatic erythrocytes to mature, normochromatic erythrocytes was observed in the high dose males at the 48 -hr sampling interval. Under the conditions of this assay, the test substance did not induce micronuclei; the test substance is negative.

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

Additional information

The test substance produced mutagenicity when evaluated in bacterial cultures, however it did not produce mutagenicity when evaluated in vivo (in animal studies). Further, the test substance was not carcinogenic in lifetime mammalian testing. 

Justification for classification or non-classification

The test substance produced mutagenicity when evaluated in bacterial cultures, however it did not produce mutagenicity when evaluated in vivo (in animal studies including an in vivo micronucleus test). Further, the test substance was not carcinogenic in lifetime mammalian testing. The substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. Although this classification deviates from the harmonized classification, the harmonized classification will be adhered as it is stricter.