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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Pigment Yellow 120 showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.

Link to relevant study records

Referenceopen allclose all

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:
From 2 MAY 2006 to 12 MAY 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 471)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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 (induced with phenobarbital/beta-naphtoflavone; experiment I); hamster liver S9 (non-induced; experiment II)
Test concentrations with justification for top dose:
Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent and its relative non-toxicity to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (TA 1535 and TA 100), 4-Nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (for all strains)
Remarks:
with metabolic activation (rat liver S9)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (TA 1535, TA 100, TA 1537, WP2 uvrA), congo red (TA 98)
Remarks:
with metabolic activation (hamster liver S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: plate incorporation assay without and with induced rat liver S9 mix (induction with phenobarbital/beta-naphthoflavone)
Experiment II: preincubation assay without and with non-induced hamster liver S9 mix

DURATION
- Preincubation period: Experiment II: 30° C for 30 minutes
- Exposure duration: at least 48 hours at 37° C

NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including the control
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent colony is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the thresshold is regarded as an indication of mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative annd solvent controls such an increase is not considered biologically relevant.
Statistics:
Arithmetic means and standard deviation of the counted colonies were calculated.
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:
cytotoxicity
Remarks:
in strain TA1537, TA98, TA100 (exp. I, without S9, starting from 2500 µg/plate); strain TA1537, TA98 (exp. II, without S9, starting from 1000 µg/plate);TA 100 exp. II, without S9, starting from 333 µg/plate); TA1537, TA98 (exp. II, with S9, 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
cytotoxicity
Remarks:
noted in exp. II, without S9 mix, at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar from 333 pg/plate up to 5000 µg/plate in experiment I. In experiment II, precipitation was observed from 100 µg/plate up to 5000 µg/plate. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment I, the data in the negative control with metabolic activation of strain WP2uvrA were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was observed in
- strain TA1537, TA98, TA100 (exp. I, without S9, starting from 2500 µg/plate);
- strain TA1537, TA98 (exp. II, without S9, starting from 1000 µg/plate);
- strain TA 100 exp. II, (without S9, starting from 333 µg/plate);
- strain TA1537, TA98 (exp. II, with S9, 5000 µg/plate) and
- E.coli strain WP2 uvrA (exp. II, without S9 mix, at 5000 µg/plate)
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results (migrated information):
negative

The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.
Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation assay. Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay without and with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the concentrations 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

Endpoint:
in vitro gene mutation study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An analogue substance was tested in a micronucleus test conducted similar to OECD guideline 474. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0, 50, 500 and 5000 mg per kg bodyweight.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test item was within the normal range of the negative control. Thenumber of normochromatic erythrocytes containing micronuclei was not increased compared to the control animals.The ratio of polychromatic/normochromatic erythrocytes in both male and femaleanimals remained unaffected by the treatment.

The positive control (Cyclophosphamide =EndoxanR) yielded positive results and thereforeindicating the sensitivity of the system.

Link to relevant study records

Referenceopen allclose all

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:
From 25 MAR 1981 to 2 APR 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given: comparable to guidelines
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, company breeding colony
- Age at study initiation: 7 to 12 weeks
- Weight at study initiation: males mean: 33 g; females mean: 26 g
- Housing: grouped (5 animals per cage) in macrolon cages (type 3) in fully airconditioned rooms
- Diet: rats/mice diet Altromin1324 (Altromin GmbH, Lage/Lippe, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2
- Humidity (%): 55+/-10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 2% starch mucilage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test compound:
Preparation of compounds dilution was done freshly each day.
6250 mg of test item were weight in a beaker, mixed with 2% starch mucilage, transferred into a 25 mL flask and topped up to the calibration mark. A solution was formed by stirring for 5 minutes on a magnetic stirrer.

Positive control:
For Endoxan(R) (= Cyclophosphamide) stock solution, 5 ml of distilled water were added to 100 mg Endoxan(R) in an injection phial and shaken to form a clear solution. The solutions for administration were prepared from this stock solution. For this purpose, 2 ml of the 2% stock solution were mixed with 6 ml distilled water.
Duration of treatment / exposure:
30 hours in total (1st application, lack phase of 24 hours, 2nd application, after 6 hours termination of test)
Frequency of treatment:
twice
Post exposure period:
6 hours
Remarks:
Doses / Concentrations:
0, 50, 500, 5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 100 mg/kg bw
Tissues and cell types examined:
polychromatic erythrocytes derived from the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
According to a preliminary study, the maximal applicable dose of the test item is 5000 mg per kg bodyweight, which was administered twice.

DETAILS OF SLIDE PREPARATION:
At the indicated time a suspension of the bone marrow of both femora was formed. The mixture was then centrifuged for 5 minutes at about 1000 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared an a cleaned slide and air-dried for about 24 hours.

Staining procedure
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts distilled water
- rinsing in distilled water
- drying with filter paper
- cleaning the backside of the slide with methanol
- 5 minutes in xylene
- coating with Entellan
Evaluation criteria:
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group to which they belonged remains unknown to the investigator.
Statistics:
The number of polychromatic erythrocytes with micronuclei occurring in the 2000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically. Increases in dose groups compared to the simultaneous control group were determined using binominal distribution. Differences in dose groups compared to the simultaneous control group were checked using the method of Nemenyi (separately for both sexes).
The statistical evaluations were performed using a in-house computer program. All statistical results are based on a 95 % level of significance.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Faeces was stained orange in all animals of dose group 500 and 5000 mg/kg bw.
Conclusions:
Interpretation of results (migrated information): negative
The results indicate that, under the conditions of the present study, the test item is not mutagenic in the micronucleus test.
Executive summary:

The test item was tested in a micronucleus test conducted similar to OECD guideline 474. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0, 50, 500 and 5000 mg per kg bodyweight.

The animals were treated twice within 24 h with the test compound and according to the test procedure the animals were killed 6 hours after the second administration of the test compound.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test item was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased compared to the control animals.The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment.

The positive control (Cyclophosphamide =EndoxanR) yielded positive results and therefore indicating the sensitivity of the system.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)
Reason / purpose:
read-across source
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

No classification - No adverse effects observed.