GOLDEN YELLOW SCJ 1006

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

FAT 40543/A is considered non-mutagenic

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:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant OECD guideline study withouit deviations

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

92/69/EEC (Micronucleus)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System: Swiss mice, CD-1 (outbred-SPF-Quality) Recommended test system in international guidelines (e.g. EPA, FDA, OECD, EEC). Females were nulliparous and non-pregnant. Source: Charles River Wiga, Sulzfeld, Germany.
Number of Animals per Test: At least 5 male and 5 female mice per sampling time in each treatment group.
Age at Start of Treatment: Approximately 7 weeks.
Body Weight at Start of Treatment: Males: 31.8 – 34.4 g/animal; Females: 25.8 – 28.0 g/animal
Identification: By unique number on the tail.
Allocation: Allocated to treatment groups as they came to hand from delivery boxes.
On arrival and prior to final assignment to study, all animals were clinically examined to ensure selected animals were in a good state of health.
ANIMAL HUSBANDRY
The animals were housed in an air-conditioned room with approximately 15 air changes per hour and a controlled environment with a temperature of 21 ±3 °C and a relative humidity of 30 - 70%. The room is illuminated with 12 hours artificial fluorescent light and 12 hours dark per day. Five animals per sex per group were housed in labelled polycarbonate cages containing purified sawdust as bedding material (Woody SPF, supplied by B.M.I., Helmond, The Netherlands). Certificates of analysis were examined and then retained in the NOTOX archives. The acclimatisation period was at least 5 days before start of treatment under laboratory conditions. The animals were provided with standard pelleted laboratory animal diet (Carfil Quality BVBV, Oud-Turnhout, Belgium) and tap-water. Certificates of analysis were examined and then retained in the NOTOX archives.

Route of administration:

other: repeated oral intubation, with a 24 hours interval

Vehicle:

1% Aqueous carboxymethyl cellulose

Details on exposure:

Method: Oral intubation
Rationale: The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.
Fasting: Feed was withheld 3 - 4 h prior to dosing until administration of FAT40543/A.
Frequency: Repeated intubation, with a 24 hours interval.
Dose Levels: Maximum tolerated (high), an intermediate and a low dose.
Dosing Volume: 10 mL/kg body weight.

Duration of treatment / exposure:

animals were dosed twice with the exeption of positive controls, which were dosed only once.

Frequency of treatment:

Twice within a 24 hours interval

Post exposure period:

24 and 48 hours following last exposure

Remarks:

Doses / Concentrations:
1000, 500 and 250 mg/kg bw
Basis:
actual ingested
by intubation

No. of animals per sex per dose:

Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 500 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 250 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 250 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 1000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 1000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 500 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 500 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 250 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 250 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

5 males and 5 females dosed with Cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta-Werke, F.R.G.) at 50 mg/kg body weight dissolved in 0.9% (w/v) NaCl (Merck) in Milli-RO water.

Tissues and cell types examined:

serum, bone marrow smears

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation at 24 and 48 h according to schedule after dosing of the vehicle, FAT40543/A and the positive control. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue) and marked (with the study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45 ° over the slide with the drop of bone marrow suspension. The preparations were then air-dried and thereafter fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.
Staining of the bone marrow smears: The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.
Analysis of the bone marrow smears for micronuclei: All slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 1000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.

Statistics:

A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combi.ned data for both sexes nor in the data for male or female groups alone.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Doses producing toxicity: P/N ratio: No effect seen Other toxic signs: No signs of toxicity observed

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean number of micronucleated polychromatic erythrocytes scored in the test substance treated groups were compared with the corresponding control groups. All animals dosed with the test substance showed no reaction to treatment. The faeces of the animals dosed with FAT40543/A were coloured reddish 20 hours after treatment. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of test substance treated animals. The incidence of micronucleated polychromatic erythrocytes in the control animals were between or equal to the minimum and maximum value of the historical control data range {male: 0 and 4 (mean 0.4)} and {female: 0 and 4 (mean 0.5)}; indicated are means for n=140. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. The groups that were treated with FAT40543/A showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls.

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that this test is valid and that FAT 40543/A is not mutagenic in the in-vivo micronucleus test under the experimental conditions described in this report.

Executive summary:

FAT 40543/A was tested in the Micronucleus Test in mice. Six groups each comprising 5 males and 5 females, received a repeated oral intubation, with a 24 hours interval. Two groups were dosed with 1000 mg/kg body weight (C and D), two groups were dosed with 500 mg/kg body weight (E and F) and two groups were dosed with 250 mg/kg body weight (G and H). Bone marrow from corresponding vehicle treated groups (A and B) served as negative controls. Bone marrow from a group (I) treated with a single oral intubation of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control. Bone marrow was sampled at 24 and 48 hours after the second dosing. Bone marrow from the positive control group (I), was harvested at 48 hours after dosing only. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with FAT 40543/A. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. The groups that were treated with FAT 40543/A showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls. It is concluded that this test is valid and that FAT 40543/A is not mutagenic in the micronucleus test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

FAT 40543/A has been investigated for mutagenic effects in three in vitro studies and one in vivo investigation. The results are summarised as follows:

In vitro Ames-Test

FAT 40543/A was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with the tryptophan-requiring strain WP2 uvrA in two independent experiments up to and including concentrations of 5000 µg/plate in the absence and presence of S9-mix. In the absence of S9-mix, FAT 40543/A induced in strain TA1537 5- and 4-fold dose-related increases in the number of revertant (His+) colonies, in experiment 1 and 2 respectively. In strain TA98 the test substance induced 7-and 6-fold dose-related increases, in experiment 1 and 2 respectively. These increases were observed only at precipitating concentrations. In the tester strains TA1535, TA100 and WP2 uvrA, FAT 40543/A did not induce a dose-related increase in the number of revertant colonies in both independent experiments. In the presence of S9-mix, FAT 40543/A induced in strain TA1535 14- and 7-fold dose-related increases in the number of revertant (His+) colonies, in experiment 1 and 2 respectively. In strain TA1537 the test substance induced 6- and 9-fold dose-related increases, in experiment 1 and 2 respectively. In TA98 the test substance induced dose related 4-fold increases in both experiments. FAT 40543/A induced in strain TA100 12- and 8-fold dose-related increases, in experiment 1 and 2 respectively. In WP2 uvrA the test substance induced dose related 2-fold increases in the number of revertant (Trp+) colonies in both experiments. The increases in tester strain TA1537 were observed only at precipitating concentrations. The increases in the four other strains were observed at non-precipitating concentrations also.

Based on the results of this study it is concluded that FAT 40543/A is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

In vitro chromosome aberrations in chines hamster lung cells

The effect of FAT 40543/A on the induction of chromosome aberrations in cultured Chinese hamster lung (CHL) cells in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix) was investigated.

In the absence of S9-mix, in the first experiment, FAT 40543/A was tested up to 33 µg/ml for a 24 h fixation time and up to 10 µg/ml for a 48 h fixation time and in the second experiment FAT 40543/A was tested up to 10 µg/ml for a 24 h fixation time, and up to 5.6 µg/ml for a 48 h fixation time.

In the presence of S9-mix FAT 40543/A was tested up to 100 µg/ml for a 24 h and 48 h fixation period in both experiments.

In the absence of S9-mix, in experiment 1, at the 24 h fixation period concentrations of 10 and 33 µg/ml and at the 48 h fixation period concentrations of 1, 3 and 10 µg/ml induced statistically and biologically significant increases in the number of cells with chromosome aberrations.

In experiment 2 at the 24 h fixation period a concentration of 10 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations when gaps were excluded. At the 48 h fixation period a concentration of 3 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations when gaps were excluded and a concentration of 5.6 µg/ml induced a statistically and biologically significant increase in the number of cells with chromosome aberrations when gaps were included and excluded.

It is concluded that the test substance is clastogenic in the absence of S9-mix.

In the presence of S9-mix, in experiment 1, at the 24 h fixation period a concentration of 33 µg/ml induced a statistically significant increase in the number of cells with chromosome aberrations. None of the other tested concentrations induced a statistically or biologically significant increase in the number of cells with chromosome aberrations.

Therefore, it is concluded that clastogenicity of FAT 40543/A is almost completely eliminated by the presence of S9-mix.

The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were optimal and that the metabolic activation system (S9-mix) functioned properly. It is concluded that the test substance is clastogenic in Chinese hamster lung (CHL) cells under the experimental conditions described in this report.

In vitro mammalian cell gene mutation test with V79 Chinese hamster cells

This report describes the effects of FAT 40543/A on the induction of forward mutations at the HPRT-locus in V79 Chinese hamster cells in the presence and absence of S9-mix.

FAT 40543/A was tested from 3 to 24.5 µg FAT 40543/A/ml in the absence of S9-mix and from 3 to 100 µg/ml in the presence of 8% (v/v) S9-fraction, in the first and second experiment respectively. In the absence of S9-mix, at these dose levels appropriate cytotoxicity was observed. FAT 40543/A did not induce a dose-related increase in the mutant frequency at the HPRT-locus in the absence and presence of S9-mix, in two independently repeated experiments. Mutant frequencies induced by positive control chemicals were increased by 42- and 32-fold for EMS, in the first and second experiment respectively, and by 19- and 21-fold for DMN, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. It is concluded that FAT 40543/A is not mutagenic in the mutation test with V79 Chinese hamster cells under the experimental conditions described in this report.

In vivo micronucleus test in mouse bone marrow cells

This report describes the effects of FAT 40543/A on the induction of forward mutations at the HPRT-locus in V79 Chinese hamster cells in the presence and absence of S9-mix.

FAT 40543/A was tested from 3 to 24.5 µg FAT 40543/A/ml in the absence of S9-mix and from 3 to 100 µg/ml in the presence of 8% (v/v) S9-fraction, in the first and second experiment respectively. In the absence of S9-mix, at these dose levels appropriate cytotoxicity was observed. FAT 40543/A did not induce a dose-related increase in the mutant frequency at the HPRT-locus in the absence and presence of S9-mix, in two independently repeated experiments. Mutant frequencies induced by positive control chemicals were increased by 42- and 32-fold for EMS, in the first and second experiment respectively, and by 19- and 21-fold for DMN, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. It is concluded that FAT 40543/A is not mutagenic in the mutation test with V79 Chinese hamster cells under the experimental conditions described in this report.

In conclusion, the in vivo test indicates the negative results although the in vitro chromosome aberration test and the Ames test indicated a positive result. The in vivo test result is more reliable for its higher similarity to the human metabolic environment. In addition, the in vitro gene mutation test in Chinese hamster cells (V79) was negative as well. Thus, we can conclude that the test substance indicates no genetic toxicity under the experimental condition.

Justification for selection of genetic toxicity endpoint
in vivo study

Justification for classification or non-classification

Based on the available results of the in vivo and in vitro tests, the substance is not classified for mutagenicity according to CLP (Regulation (EC) No 1272/2008) or DSD (Directive 67/548/EEC) respectively.