Salicylamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative, Ames test, S. typhimurium TA1535, TA100, TA1538, TA98, TA1537; E. coli K12/343/113, King 1979

Negative, Ames test, S. typhimurium TA98, TA100; Kuboyama 1992

Negative, Ames test, S. typhimurium TA100, TA1535, TA97, TA98; Zeiger 1992

Positive, chromosome aberration test - in vitro, CHL cells, Ishidate 1988

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study published in a peer-reviewed journal, according to scientific standards. Experimental details well documented, no explicit reference to guideline. Strains tested: S. typhimurium TA1535, TA100, TA1538, TA98, TA1537; E. coli K12/343/113. The strains TA102 (S. typhimurium) or WP2 uvrA (E. coli) requested by the guideline OECD 471 were not tested. Highest tested dose 9600 micrograms/plate with S. Typhimurium, but only 3700 micrograms/plate with E.coli. No individual data on treated plates and positive/negative controls reported; significance of differences in mutant frequencies assessed by chi square test.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Strains TA102 and/or WP2 uvrA lacking; E.coli: tested only up to 3600 microgram/plate

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his G46, D3052, C3076
E. coli K12/343/113: mutations to 5-methyltryptophane resistance, galactose utilization, arginine independence tested

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

E. coli, other: K12/343/113

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fraction (Sprague-Dawley SIV-50 rats) induced with Aroclor 1254

Test concentrations with justification for top dose:

generally: at least 5 concentrations up to 3600 microgram/plate
highest tested dose from table of experimental results: S. typhimurium 70 micromoles/plate = 9600 microgram/plate; E.coli: 10 mM = 3600 microgram/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO 30 microliter
- Justification for choice of solvent/vehicle: solubility of test substance

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), 2-aminoanthracene, N-nitrosomorpholine

Details on test system and experimental conditions:

METHOD OF APPLICATION: S. typhimurium: in agar (plate incorporation); sometimes also with preincubation; E. coli: in suspension

DURATION
- Preincubation period: 30 min at 37°C
- Exposure duration: not explicitly stated (routine test according to Ames BN et al, Mutation Research 31: 347-364 (1975))

NUMBER OF REPLICATIONS: no explicit data (sufficient to perform chi square test)

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- No details given
- Probably none observed, since concentrations up to 9600 micrograms/plate were reported

Statistics:

Chi square test to assess significance of differences between absolute mutant frequencies in the control and the test series

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

9600 micrograms/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

probably none, max. dose 9600 micrograms/plate reported

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 9600 micrograms/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

probably none, max. dose 9600 micrograms/plate reported

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

E. coli, other: K12/343/113

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 3600 micrograms/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

probably none, max. dose 3600 micrograms/plate reported

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Intrasanguineous host mediated test (Mohn G, Ellenberger J, The use of Escherichia coli K12/343/113 as a multipurpose indicator strain in various mutagenicity testing procedures, in Kilbey M, Legator W, Nichols W & Ramel C, Handbook of mutagenicity test procedures, Elsevier Amsterdam (1977), 95 -118) in female NMRI mice injected with 8000'000'000 cells E. coli K12/343/113 and treated with salicylamide, 550 mg/kg i.p.:

Negative

Conclusions:

Interpretation of results (migrated information):
negative

The study is considered to be reliable with some restrictions and suitable for use as part of a weight-of-evidence assessment.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study published in a peer-reviewed journal, according to scientific standards. Experimental details well documented, no explicit reference to guideline. Strains tested: S. typhimurium TA98 and TA100. The strains TA 1535, TA1537, TA102 (S. typhimurium) or WP2 uvrA (E. coli) requested by the guideline OECD 471 were not tested. Highest tested dose 100 micrograms/plate.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only strains TA98 and TA100; maximum dose 100 microgram/plate

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his D3052, G46

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 liver mix from male rats (SD strain), male mice (ddy strain), male guinea pigs (Hartley strain) and male golden hamsters; all induced with 500 mg/kg polychlorobiphenyl i.p.

Test concentrations with justification for top dose:

0.1 mg / plate

Vehicle / solvent:

DMSO 0.1 ml / plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: AF-2 (furyl furamide, 2-(furan-2-yl)-3-(5-nitrofuran-2-yl)prop-2-enamide), benzopyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 min at 37°
- Exposure duration: 48 h at 37°

NUMBER OF REPLICATIONS: 5

NUMBER OF CELLS EVALUATED: no data

Evaluation criteria:

number of his+ revertants more than twice the number of spontaneous revertants considered to be mutagenic

Statistics:

mean and standard deviation for 5 plates

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Most revertant numbers below those of DMSO control

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Maximum ratio of revertant numbers (treated / DMSO control) = 1.27

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The study is considered to be reliable with some restrictions and suitable for use as part of a weight-of-evidence assessment.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study published in a peer-reviewed journal, according to scientific standards, within the National Toxicology Program's mutagenicity program. Two independent tests were performed at different laboratories.Experimental details well documented, no explicit reference to guideline. Strains tested: S. typhimurium TA100, TA1535, TA97, TA98. The strains TA102 (S. typhimurium) or WP2 uvrA (E. coli) requested by the guideline OECD 471 were not tested.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Strains TA102 and/or WP2 uvrA lacking

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his G46, C3076, D3052

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

10% and 30% S-9 fraction of male Syrian hamster liver, Aroclor 1254 induced, 10% and 30% S-9 fraction of male Sprague-Dawley rat liver, Aroclor 1254 induced

Test concentrations with justification for top dose:

Laboratory MIC (Microbiological Associates, Rockville MD): 100, 333, 1000, 3334, 6667 microgram/plate
Laboratory SRI (SRI International, Menlo Park, CA): 33, 100, 333, 1000, 1666, 3334 microgram/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of test substance

Untreated negative controls:

yes

Remarks:

water, in test for acetic acid and others

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Migrated to IUCLID6: 9-aminoacridine, 4-nitro-o-phenylenediamine

Untreated negative controls:

yes

Remarks:

water, in test for acetic acid and others

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min. at 37°C
- Exposure duration: 2 days at 37°C

NUMBER OF REPLICATIONS:
- triplicate per trial and dose
- repeat trial at least one week following the initial trial

NUMBER OF CELLS EVALUATED:
- no data
- plates machine-counted (New Brunswick; Artek) unless precipitate present

DETERMINATION OF CYTOTOXICITY
- Method: Initial toxicity assay with TA100; toxic concentrations defined as those producing a decrease in number of his+ colonies, a clearing of the density of the background lawn, or both

Evaluation criteria:

Individual trials judged mutagenic (+), weakly mutagenic (+W), questionable (?), or non-mutagenic (-), depending on magnitude of increase in his+ revertants, and the shape of the dose-response. Questionable (?) applied, if only a single dose elevated, or a weak increase not dose-related. It was not necessary for a response to reach two-fold over background to be judged mutagenic.
A chemical was judged mutagenic, if it produced a reproducible, dose-related response over solvent control in replicate trial, under a single metabolic activation condition.

Statistics:

Mean and SEM (standard error of mean)

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight clearing of background lawn at 6667 microgram/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight clearing of background lawn at 6667 microgram/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight clearing of background lawn at 6667 microgram/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Good consistence between results from two laboratories. Maximum increase of revertant count over solvent control in any single dose: 30 % (TA 100, 30% hamster liver S-9, in only one laboratory). No dose-related increase in any trial.

Conclusions:

Interpretation of results (migrated information):
negative

The study is considered to be reliable with some restrictions and suitable for use as part of a weight-of-evidence assessment.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study published in a data book by the National Institute of Hygienic Sciences, Tokyo, Japan, containing chromosome aberration data on 779 chemicals. Study performed according to scientific standards, experimental details well documented, explicit reference to guidelines OECD 473 and US EPA OPPTS "In vitro mammalian cytogenetics". Deficiencies: No replications indicated; only 100 metaphases per concentration counted. Positive results for salicylamide (including polyploidy) were only observed at a cytotoxic concentration (>50% inhibition) of 3.6 mM. A secondary source (Ishidate et al, Mutation Research 195: 151-213 (1988) quotes the original result without any further information.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 1983

Deviations:

yes

Remarks:

No replications indicated, only 100 metaphases per concentration counted

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1982

Deviations:

yes

Remarks:

No replications indicated, only 100 metaphases per concentration counted

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Fibroblast cell line from the lung of a newborn female Chinese hamster, established in 1970
- Type and identity of media: Eagle's MEM (Gibco) with 10% inactivated calf serum
- Properly maintained: yes (single-cell subclone 1973-1987)
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: yes (25 chromosomes, compared with 22 for Chinese hamster)
- Periodically "cleansed" against high spontaneous background: no data
- Doubling time: approx. 15 hours
- Cells proliferate in monolayers

Metabolic activation:

with and without

Metabolic activation system:

Rat (Wistar or Fisher) liver S9 fraction, after induction with 500 mg/kg polychlorinated biphenyls i.p.

Test concentrations with justification for top dose:

0.125, 0.25, 0.5 mg/ml

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

414/779 substances tested negative

Positive controls:

yes

Remarks:

313/779 substances tested positive

Remarks:

positive controls not explicitly mentioned as such

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 3 days
- Exposure duration: 24 and 48 hours
- Expression time (cells in growth medium): 24 and 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hours

NUMBER OF REPLICATIONS:
- none mentioned

NUMBER OF CELLS EVALUATED: 100 metaphases

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth: Cell proliferation after 2 days of exposure assessed indirectly by rate of staining (0.1% crystal violet after formalin fixation), proliferation of solvent control set to 100%. A concentration with approximately 50% inhibition was used as standard.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no data

Evaluation criteria:

Structural aberrations classified as chromatid gaps, breaks, and exchanges, or chromosome gaps, breaks, and exchanges. A cell having any of these was recorded as aberrant.
Negative: <5% aberrant cells, inconclusive 5 - <10%, positive 10% and more

Statistics:

Least-square regression lines incl. solvent control points (linear or semi-logarithmic) used to interpolate or extrapolate to D20 (concentration in mg/ml inducing any aberration in 20% of the metaphases).
TR values in experiments with positive results were calculated as E/D (with E= number of cells with exchanges, D = concentration in mg/ml). The highest TR value was reported for the substance.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

10% incidence of aberrant cells (exactly at threshold) after 48 hours; 7% after 24 hours (inconclusive)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

It must be noted that gaps are included in the score of cells with structural chromosome aberrations, although they should generally not be included in the total aberration frequency, according to OECD 473. Their frequency is 3% (of a total of 7%) after 24 hours, and 2% (of a total of 10%) after 48 hours.

Detailed results show that chromosome aberration occurs only at the highest concentration (3.6 mM); the frequency is 7% (inconclusive) after 24 hours and just reaches the margin of "positive" (10%) after 48 hours. Polyploidy also occurs only at the highest concentration.

Sample selection in this study was based on cytotoxicity preliminary screening; a concentration with approx. 50 % inhibition was used as a standard, and usually 3 concentrations above and below this standard were included in the CA test. In case of no cytotoxicity , about 10 mM were tested.

No details on cytotoxicity are reported for salicylamide, but from the highest tested concentration of 0.5 mg/mL = 3.6 mM we conclude that

1. there was cytotoxicity observed (without cytotox, the testing limit would be about 10 mM)

2. the second highest concentration tested (0.25 mg/mL) would be the standard with approximately 50% inhibition, and the highest concentration was probably more inhibitory.

It cannot be derived from the data whether a high degree of cytotoxicity influenced the results of the chromosome aberration test.

Treatment time	Concen- tration	Meta- phases	Polyploid			Chromatid changes %			Chromo- some changes %			Judge- ment
hrs	mg/mL	number	%	Judge	Gaps	Breaks	Exch.	Gaps	Breaks	Exch.	Total
24	None	100	1		2	0	0	0	0	0	2
24	0.125	100	0	-	3	0	0	0	0	0	3	-
24	0.25	100	2	-	3	1	0	0	0	0	4	-
24	0.5	100	1	-	3	5	0	0	0	0	7	+/-
48	Ethanol	100	0		0	0	1	0	0	0	1
48	0.125	100	1	-	0	0	0	0	0	0	0	-
48	0.25	100	4	-	1	1	2	0	0	0	3	-
48	0.5	100	12	+	2	7	3	0	0	0	10	+

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation after 24 hours: ambiguous (7% aberrations), after 48 hours: positive (10% aberrations, including gaps, borderline)

The study is considered to be reliable with some restrictions and suitable for use as part of a weight-of-evidence assessment. The result is considered to be borderline.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Negative, sister chromatid exchange - in vivo, mouse, Giri 1996

Negative, chromosome aberration test - in vivo, mouse, Giri 1996

Negative, micronucleus test - in vivo, mouse, King 1979

Negative, sex-linked recessive lethal test, drosophila, King 1979

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study published in a peer-reviewed journal, according to scientific standards. Experimental details well documented, no explicit reference to guideline.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

Housing temperature (28+-2°C) too high. Only male mice tested (5 per dose) - no justification given.

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)

Deviations:

yes

Remarks:

Only male mice tested (4 per dose i.p., 5 for oral gavage----) - no justification given.

GLP compliance:

not specified

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Division of Laboratory animals, Central Drug Res. Institute, Lucknow, India
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 30 g
- Assigned to test groups randomly: no data
- Fasting period before study: no
- Housing: five per cage, husk bedding
- Diet: Standard rodent pellet diet, Gold Mohor, Lipton India Ltd, Chandigarh, India (ad libitum)
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 28 +/- 2 °C
- Humidity (%): 60 +/- 5 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12 hours

IN-LIFE DATES: no data

Route of administration:

other: intraperitoneal (3 dose levels); or oral (gavage) 1 dose level

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO (for i.p. application), suspension in 2% gum acacia in distilled water (for gavage application)
- Justification for choice of solvent/vehicle: solubility of the test substance
- Concentration of test material in vehicle: dose-dependent: approx. 20, 40, and 80 mg/mL (i.p.), 35 mg/mL (gavage)
- Amount of vehicle (if gavage or dermal): DMSO 75 microliter/mouse for i.p., 2% gum acacia in water 0.3 mL per mouse for gavage
- Type and concentration of dispersant aid (if powder): 2% gum acacia
- Lot/batch no. (if required): no data
- Purity: no data

Duration of treatment / exposure:

i.p. administration: 24 hours, gavage administration: 24 hours
colchicine: 2 hours before sacrifice

Frequency of treatment:

single treatment

Post exposure period:

none (time between administration and sacrifice: i.p. 24 hours, gavage 24 hours)0

Remarks:

Doses / Concentrations:
i.p. 50, 100, 200 mg/kg body weight
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
oral (gavage) 350 mg/kg body weight
Basis:
nominal conc.

No. of animals per sex per dose:

4 males per dose, i.p. administration
5 males, oral administration

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide:
- Justification for choice of positive control(s): no data
- Route of administration: i.p. only
- Doses / concentrations: 25 mg/kg

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
i.p.: based on the results of the Sister Chromatid Exchange (SCE) dose-response study: one higher dose was selected, since chromosome aberration is less sensitive than SCE
oral/gavage: 1/3 of approx. oral LD50

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- i.p. injection and gavage 22 h before colchicine, 24 hours before sacrifice
- colchicine (2 mg/kg) 2 h before sacrifice
- sacrifice and expelling of bone marrow 24 hours after treatment

DETAILS OF SLIDE PREPARATION:
- bone marrow chromosomes prepared according to Preston RJ et al (1987) Mutation Res. 198: 157-165
- staining of chromosomes on slide with Giemsa

METHOD OF ANALYSIS:
- 100 well-spread metaphase cells scored per animal
- mitotic indices (MI) calculated from 1000 cells / animal, expressed as percentage
- chromosome aberrations (CA) scored according to WHO method (Preston et al, see above)
- aberration frequencies of chromatid and chromosome type per cell calculated
- gaps recorded but not included in the frequency of aberrations per cell

Evaluation criteria:

Significant increase in chromosome aberration
Gaps recorded but not included in the frequency of aberrations per cell

Statistics:

Statistical calculations carried out from percentages of aberrant cells
Student's t-test, to compare results at each dose with control

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

no mortality

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

No significant increase in aberrations / cell and aberrant cells (%) for all doses tested, when compared to solvent control.

No significant difference in mitotic indices, when compared to control.

Conclusions:

Interpretation of results (migrated information): negative

Reference 2

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study published in a peer-reviewed journal, according to scientific standards. Experimental details well documented, no explicit reference to guideline. Only four animals / dose, male and female (number per sex not specified). No information on housing conditions, diet.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Only 4 mice per dose

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Supplier:S. Ivanovas GmbH, Kisslegg, Germany
- Weight at study initiation: about 30 g
- Fasting period before study: no data
- Housing: no data
- Diet / Water / Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C) / Humidity (%) / Air changes (per hr) / Photoperiod (hrs dark / hrs light): no data

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: not precisely specified: probably dispersion in 3% gum arabic (the two alternative solvents stated in the article, 0.9% NaCl and olive oil, are unlikely to dissolve the test substance)

Duration of treatment / exposure:

30 hours (two i.p. injections 24 hours apart, sacrifice 6 hours after the second injection)

Frequency of treatment:

Twice

Post exposure period:

6 hours after the second injection

Remarks:

Doses / Concentrations:
5 mmoles/kg, 685 mg/kg
Basis:
nominal conc.
Highest tested dose

No. of animals per sex per dose:

usually 4 mice, number per sex not specified

Control animals:

yes

Positive control(s):

no specific data (various mutagens were administered, representative data reported in Wild D (1978) Mutation Res. 56: 319-327)

Tissues and cell types examined:

Bone marrow smears; polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- non-toxic to approximately lethal, based on previous toxicity experiments

DETAILS OF SLIDE PREPARATION:
- bone-marrow smears, stained with May-Gruenwald and Giemsa stains

METHOD OF ANALYSIS:
- 1000 polychromatic erythrocytes analyzed per animal

Evaluation criteria:

According to Schmid W (1976) in Hollaender A, Ed., Chemical Mutagens, Vol 4: 31-53, Plenum New York

Statistics:

Calculation of significance of results according to tables by Kastenbaum MA & Bowman KO (1976), Mutation Res. 9: 527-549

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

other: control group mentioned, but no data given

Negative controls validity:

not specified

Positive controls validity:

other: control groups with various mutagens mentioned, but no data given

Conclusions:

Interpretation of results (migrated information): negative

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study published in a peer-reviewed journal, according to scientific standards. Experimental details well documented, no OECD guideline, no explicit reference to EPA OPPTS 870.5915.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5915 (In Vivo Sister Chromatid Exchange Assay)

Deviations:

yes

Remarks:

BrdU tablets implanted 1 h before (not after) test substance administration. Only male mice tested (5 per dose) - no justification given. Room temperature of animal facilities unusually high (28°C).

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Division of Laboratory animals, Central Drug Res. Institute, Lucknow, India
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 30 g
- Assigned to test groups randomly: no data
- Fasting period before study: no
- Housing: five per cage, husk bedding
- Diet: Standard rodent pellet diet, Gold Mohor, Lipton India Ltd, Chandigarh, India (ad libitum)
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 28 +/- 2 °C
- Humidity (%): 60 +/- 5 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12 hours

IN-LIFE DATES: no data

Route of administration:

other: intraperitoneal (3 dose levels); or oral (gavage) 1 dose level

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO (for i.p. application), suspension in 2% gum acacia in distilled water (for gavage application)
- Justification for choice of solvent/vehicle: solubility of the test substance
- Concentration of test material in vehicle: dose-dependent: approx. 10, 20, 40 mg/mL (i.p.), 35 mg/mL (gavage)
- Amount of vehicle (if gavage or dermal): DMSO 75 microliter/mouse for i.p., 2% gum acacia in water 0.3 mL per mouse for gavage
- Type and concentration of dispersant aid (if powder): 2% gum acacia
- Lot/batch no. (if required): no data
- Purity: no data

Duration of treatment / exposure:

i.p. administration: 23 hours, gavage administration: 23.5 hours
BrdU: 24 hours before sacrifice
colchicine: 2 hours before sacrifice

Frequency of treatment:

single treatment

Post exposure period:

none (time between administration and sacrifice: i.p. 23 hours, gavage 23.5 hours

Remarks:

Doses / Concentrations:
i.p. 25, 50, 100 mg/kg body weight
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
oral (gavage) 350 mg/kg body weight
Basis:
nominal conc.

No. of animals per sex per dose:

5 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Justification for choice of positive control(s): Preston RJ et al (1987) Mutation Res. 198: 157-165
- Route of administration: i.p. only
- Doses / concentrations: 1.5 mg/kg

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
i.p.: 1/10 of approx. oral LD50, since no i.p. LD50 available
oral/gavage: 1/3 of approx. oral LD50

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- BrdU tablet (paraffin-coated, 80% of surface) implanted subcutaneously under anesthesia
- i.p. injection 1 h after tablet implantation, or gavage 1/2 h after tablet implantation
- colchicine (4 mg/kg) 22 h after tablet implantation
- sacrifice and expelling of bone marrow 2 hours later, i.e. 24 hours after tablet implantation

DETAILS OF SLIDE PREPARATION:
- hypotonic treatment: 20 min, 0.075 M KCl at 37°
- three times fixing with methanol / acetic acid 3:1
- differential staining of chromosomes on slide with fluorescence-plus-Giemsa technique

METHOD OF ANALYSIS:
- 30 second division metaphase cells (40 +/- 2 chromosomes) per animal scored for sister chromatid exchange SCE
- this is a total of 150 cells per dose scored
- randomly selected metaphase cells scored for replicative indices RI by staining pattern as first (M1), second (M2) and third (M3) division metaphases
- RI = (1*M1 + 2*M2 + 3*M3) / 100

Evaluation criteria:

Significant increase of SCE over control
dose-related increase
change in replicative index

Statistics:

Student's t-test, to compare results at each dose with control

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

no mortality

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

No significant increase in sister chromatid exchange (SCE) for all doses tested, when compared to solvent control.

No significant differences in replicative index (RI), when compared to control.

Conclusions:

Interpretation of results (migrated information): negative

Reference 4

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study published in a peer-reviewed journal, according to scientific standards. Experimental details well documented, no explicit reference to guideline. DMSO (2% in glucose solutuion) used as vehicle. No individual data on the number of chromosomes tested, the number of nonfertile males and the number of lethal chromosomes per exposure concentration and mating period. No confirmatory second experiment reported.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)

Deviations:

yes

Remarks:

No individual data reported

GLP compliance:

not specified

Type of assay:

Drosophila SLRL assay

Species:

Drosophila melanogaster

Strain:

other: Berlin K (wild type) males, Basc females (In(1)sc S1L sc 8R + S, sc S1 sc 8 w a B)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Males Berlin K, age 1-2 days

Route of administration:

oral: feed

Vehicle:

5% sucrose solution containing 2% DMSO

Duration of treatment / exposure:

3 days

Frequency of treatment:

continuous feeding

Post exposure period:

3 broods, each of 3 days duration

Remarks:

Doses / Concentrations:
20 mM = 2740 mg/L
Basis:
nominal in diet
Highest tested dose

No. of animals per sex per dose:

> 1000 F1 females per brood

Control animals:

yes, historical

Positive control(s):

no data

Tissues and cell types examined:

Sex-linked recessive lethal chromosomes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- maximally tolerated dose (up to the LD50)

Evaluation criteria:

Sex-linked recessive lethals scored in F2 generation and confirmed in F3 generation

Statistics:

Probability of significance , by test of Kastenbaum MA & Bowman KO (1976), Mutation Res. 9: 527-549

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No individual data on the number of chromosomes tested, the number of nonfertile males and the number of lethal chromosomes per exposure concentration and mating period. However, for the only positive test substance in this article, 1,2-dichloroethane, the number of X-chromosomes tested, the number of lethals, the recessive lethal mutation (%), and the probability of significance are tabulated per brood. It is very likely that the same data were also collected for the negative test substances, although they are not shown in the publication.

Conclusions:

Interpretation of results (migrated information): negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames test (bacterial reverse mutation in vitro): Negative

The study by King (1979) is probably the best-documented report on Ames testing, with the maximum number of strains tested (S. typhimurium TA1535, TA100, TA1538, TA98, TA1537; E. coli K12/343/113), which all show negative results, with and without metabolic activation. However, being published in 1979, it provides no data on E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102, as requested by newer versions of the guideline OECD 471. It is known that the strains tested by King may not detect certain oxidising mutagens, cross-linking agents and hydrazines, but since the test substance salicylamide has neither of these properties, we believe that the missing strain does not disqualify the negative result obtained by King.

The study by Zeiger (1992) is also well-documented and shows negative results for S. typhimurium TA100, TA1535, TA98, and additionally TA97, which supports the King result, as does the study by Kuboyama (1991), who tested only S. typhimurium TA98 and TA100, both negative with and without metabolic activation. The second paper by Zeiger (1996) is probably a quotation of their 1992 results.

Based on a weight-of-evidence approach, according to Regulation (EC) No. 1907/2006, Annex XI, section 1.2., it is concluded that salicylamide is non-mutagenic in the Ames test.

Chromosome aberration test in vitro: Positive

Ishidate (1988, Data Book) reports a large number of chromosome aberration (CA) tests (for 779 substances) and explicitly refers to guidelines OECD 473 and EPA OPPTS "In-vitro mammalian cytogenetics". Ishidate (1988, Mutation research, 951 substances), refers to the same experimental data. A positive chromosome aberration result is reported for salicylamide, albeit with the following caveats:

- The number of metaphases evaluated is only 100, although the present-day OECD guideline asks for at least 200 well-spread metaphases.

- Gaps (3 out of 7% after 24 h, 2 out of 10 % after 48 h) are counted as chromosomal aberrations, although OECD 473 advises: Gaps or achromatic lesions are recorded separately and not included in the total aberration frequency.

- The result at the usual screening harvest time (24 hours) is ambiguous (7% cells with structural CAs, thereof 3% gaps).

- The 48-hour result is just at the edge of the "positive" definition (10% or more): 10% CA, thereof 2% gaps. If gaps were not counted, the result would be "ambiguous".

- The middle concentration, 0.25 mg/mL, intended to cause approximately 50% inhibition of cell proliferation, yields negative results at 24 h (4% CA, thereof 3% gaps) and 48 h (3% CA, thereof 1% gaps). An ambiguous or positive effect is only seen at 0.5 mg/mL, which is assumed to be above the 50% inhibitory level. Although not numerically specified, a relatively high cytotoxicity may have interfered with normal metaphase formation.

Although this study reports a positive result in vitro, it is overrun by negative results in vivo (section 7.6.2, Giri 1996: chromosome aberration, and King 1979: mouse micronucleus test).

Other in vitro tests: Inconclusive

Kuboyama (1992) reports a negative Rec-assay (Bacillus subtilis, strains H17 and M45). Since there is no guideline for this type of test, this was only considered supporting information for the conclusion that salicylamide is not mutagenic in bacterial test systems.

Vitotox assay (Westerink 2009), which is a bacterial reporter screen in S. typhimurium designed as a faster alternative to the Ames test, but not yet validated, gave a positive result for salicylamide (with metabolic activation, lowest, and only, effective concentration 0.1 mM). The same article lists a positve result in an Ames test, without giving a source or any further detail. Since the Vitotox test is not validated, and the Ames result not documented, and both contradict all other Ames results quoted above, it was decided to disregard this study.

RadarScreen assay (Westerink 2009), a yeast reporter screen designed as a faster alternative to established clastogenicity tests, but not yet validated and accepted by authorities, gave a negative result for salicylamide. The positive chromosome aberration result listed in the same paper is correctly quoted from Ishidate (1988).

Additional information from genetic toxicity in vivo:

In vivo tests: Negative

All in-vivo studies were considered key studies, since they were published in peer-reviewed journals, were well-documented, and followed pertinent guidelines with only minor deviations.

Sister chromatid exchange in vivo (Giri 1996) gave a negative result in the mouse.

Sex-linked recessive lethal test (King 1979) also produced a negative result (Drosophila melanogaster).

The two in vivo tests recommended (in case of positive in-vitro mutagenicity findings) to decide whether a genotoxic potential can be expressed in vivo, yielded clear negative results:

A rodent (mouse) bone marrow clastogenicity study (chromosome aberration in vivo, Giri 1996) showed no genotoxicity for salicylamide.

A mouse bone marrow micronucleus test in vivo (King 1979) also gave a negative result.

Since salicylamide is used as an analgetic, and has proven pharmacological effects in mice and rats, there is no doubt as to its bioavailability.

Therefore the results of the four in vivo tests indicate that salicylamide is not genotoxic in vivo.

Justification for selection of genetic toxicity endpoint
The two in vivo tests recommended (in case of positive in-vitro mutagenicity findings) to decide whether a genotoxic potential can be expressed in vivo, yielded clear negative results:
A rodent (mouse) bone marrow clastogenicity study (chromosome aberration in vivo, Giri 1996) showed no genotoxicity for salicylamide.
A mouse bone marrow micronucleus test in vivo (King 1979) also gave a negative result.

Justification for classification or non-classification

The substance demonstrated negative results in several in vitro bacterial gene mutation tests (Ames), and tested positive in a single in vitro chromosome aberration test with mammalian cells. A positive result in only one in vitro mutagenicity assay is generally not sufficient to lead to classification for mutagenicity. Furthermore, four in vivo mutagenicity tests, including somatic cell bone-marrow metaphase analysis and micronucleus tests, gave consistently negative results. As a result, and in accordance with Regulation (EC) No. 1272/2008, Annex I, Part 3, 3.5.2, salicylamide is not considered to be classified for germ cell mutagenicity.