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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Not mutagenic
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 23rd to June 11th, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test conducted according to internationally accepted Guideline and in according to the GLP Principles
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK+/- mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518). The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination.
Permanent stocks of the L5178Y TK+/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.Prior to use cells were clensed of pre-existing mutants. Cultures of the cells are grown in RPMI 1640 minimal medium supplemented with 10 % horse serum heat-inactivated at 56 °C for 20 minutes before use (Complete medium 10 %). The incubations are at 37 °C in a 5 % carbon dioxide atmosphere (100 % nominal relative humidity).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 liver tissue fraction
Test concentrations with justification for top dose:
Assay n. 1:
S9 mix: (-/+);
Treatement time: 3 hours;
Dose levels: 625, 198, 62.5, 19.8 and 6.25 µg/ml

Assay n. 2:
S9 mix: (-);
Treatement time: 24 hours;
Dose levels: 625, 313, 156, 78.1, 39.1 and 19.5 µg/ml

Assay n. 2:
S9 mix: (+);
Treatement time: 3 hours;
Dose levels: 625, 313, 156, 78.1 and 39.1
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the substance is soluble in DMS up to the maximum concentration tested (625 µg/ml)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methylmethanesulphonate (absence of S9 metabolism) batch no.: 76296KJ obtained from Sigma Chemical Co.; benzo(a)pyrene (presence of S9 metabolism) batch no.: 129K1892 obtained from Sigma Chemical Co.
Details on test system and experimental conditions:
METHOD OF APPLICATION:

DURATION
- Incubation: at 37 °C in a 5 % carbon dioxide atmosphere (100 % nominal relative humidity).
- Exposure duration: 3 and 24 hours.
- Expression time (cells in growth medium): During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2 x 10^5 cells/ml.

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 2x10^5 cells/ml
Evaluation criteria:
induced mutant frequency
Statistics:
Statistical analyses will be performed according to UKEMS guidelines
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: see below for further information
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

CYTOTOXICITY TEST

Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 625 μg/ml and at a wide range of lower dose levels: 313, 156, 78.1, 39.1, 19.5, 9.77, 4.88 and 2.44 μg/ml. Upon addition of the test item to the cultures, precipitate was noted at 625 and 313 μg/ml. At the end of the 3 hour treatment period, precipitate was observed at the same dose levels and opacity of the treatment medium was observed at 156 and 78.1 μg/ml. At the end of the 24 hour treatment period, precipitate was observed at 625 μg/mL and opacity was observed at 313 and 156 μg/ml.

In the absence of S9 metabolic activation, using the 3 hour treatment time,dose related toxicity was observed at the three highest dose levels reducing relative survival (RS) to 30 %, 50 % and 65 % of the concurrent negative control value, while no toxicity was noted over the remaining concentrations tested. Using the 24 hour treatment time, dose related toxicity was observed at the three highest dose levels reducing relative survival (RS) to 23 %, 28 % and 79 % of the concurrent negative control value. No toxicity was observed over the remaining dose levels tested. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), slight toxicity was observed at the five highest dose levels. No toxicity was observed over the remaining dose levels tested.

 

MUTATION ASSAYS

Results obtained are presented in the following table:

 

Assay No.

S9

Growth Factor

Cloning efficiency (%)

1

-

15

119

2

-

24

111

 

The study was accepted as valid.

 

Survival after treatment

In the first assay, in the absence of S9 metabolic activation, dose related toxicity was observed: the RTG was reduced to 29 % at the highest dose level compared to the concurrent negative control value, and was reduced to 42 % and 68 % at the two lower dose levels. In the presence of S9 metabolic activation, slight toxicity was observed at the highest concentration.

In the second assay, using a long treatment time in the absence of S9 metabolic activation, toxicity from slight to marked was observed at the three highest dose levels. The RTG was reduced to 19 %, 32 % and 73 % compared to the concurrent negative control value. In the presence of S9 metabolic activation, toxicity was observed at the dose level of 313mg/ml and not at 625mg/ml; this event is probably due to the different solubilisation of the test item in the treatment medium (presence of precipitate).

 

Mutation results

No increases in mutant frequency were observed in the absence or presence of S9 metabolic activation, following treatment with the test item at any concentration level. For the negative and positive controls, the number of wells containing small colonies and those containing large colonies were reported. The small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. An adequate recovery of small colony mutants was observed following treatment with the positive controls.

 

OSMOLALITY AND pH

The pH values and osmolality of the post-treatment media were determined The addition of the test item solution did not have any obvious effect on the osmolality of the treatment medium.

A slight decrease of pH was observed at all dose levels tested in the absence of S9 metabolism. However, since this decrease was within the physiological values, it is not considered to affect the test system.

Conclusions:
Interpretation of results (migrated information):
negative

The substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro, in the absence or presence of S9 metabolic activation, under the experimental conditions.
Executive summary:

Method

The test item AP 1300 S was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells afterin vitrotreatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

 

Observation

AP 1300 S was found to be soluble in dimethylsulfoxide (DMSO) at the concentration of 62.5 mg/ml, corresponding to 625 μg/ml in the final treatment medium. On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation,

the test item was assayed at a maximum dose level of 625 μg/m and at a wide range of lower dose levels: 313, 156, 78.1, 39.1, 19.5, 9.77, 4.88 and 2.44 μg/ml.

In the absence of S9 metabolic activation, using the 3 hour treatment time, dose related toxicity was observed at the three highest dose levels reducing relative survival (RS) to 30 %, 50 % and 65 % of the concurrent negative control value. Using the 24 hour treatment time, dose related toxicity was observed at the three highest dose levels reducing relative survival (RS) to 23 %, 28 %

and 79 % of the concurrent negative control value. No toxicity was observed over the remaining dose levels tested.

Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), slight toxicity was observed at higher dose levels. By the end of treatment time, precipitation of the test item or opacity of the treatment medium were observed at the four or three highest dose levels.

Based on the results obtained in the preliminary trial, two independent assays for mutation to trifluorothymidine resistance were performed using the dose levels below:

 

Assay n. 1:

S9 mix: (-/+);

Treatement time: 3 hours;

Dose levels: 625, 198, 62.5, 19.8 and 6.25 µg/ml

 

Assay n. 2:

S9 mix: (-);

Treatement time: 24 hours;

Dose levels: 625, 313, 156, 78.1, 39.1 and 19.5 µg/ml

 

Assay n. 2:

S9 mix: (+);

Treatement time: 3 hours;

Dose levels: 625, 313, 156, 78.1 and 39.1

 

The dose levels of the second experiment in the presence of S9 metabolism were selected on the basis of the results of the first experiment in order to focus on the highest concentrations that could be tested.

 

Results

No increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism.

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

Additional information

Additional information from genetic toxicity in vitro:

According to the REACH Regulation (EC n. 1907/2006) Annex VII, VII and IX, Column 1, the mutagenicity potential has to be tested by means of:

Annex VII:

-In vitro gene mutation study in bacteria

Annex VIII:

-In vitro cytogenicity study in mammalian cells or in vitro micronucleus study

-In vitro gene mutation study in mammalian cells if a negative result in Annex VII, section 8.4.1 and Annex VIII, section 8.4.1.

Annex IX:

-In vivo somatic cell genotoxicity study.

 

IN VITRO GENE MUTATION STUDY IN BACTERIA

No test on the substance in itself is available, nevertheless four studies on the structural analogous Similar Substance 01 (CAS 21580-44-2) and on the precursor/metabolite Similar Substance 02 (CAS 79-94-7) have been considered in order to complete the assessment due to the great similarity with AP 1300 S.

Tests conducted on Similar Substance 01

The first study has been reported on NTP Database and the substance showed no evidence of mutagenicity in the tested S. typhimurium strains.

The second one is reported from Brusick D. (1982)[1] in the WHO publication[2] and indicates that the tested substance is a direct-acting mutagen and that a rat liver S9 mix converts the test material to a less mutagenic form.

Tests conducted on Similar Substance 02

Both the first and the second studies have been reported on a NTP Report.

During the first test the substance has been tested in 5 different concentrations on S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without S9 mix. The tested substance showed no evidence of mutagenicity in S. typhimurium strains TA98, TA100, TA1535, or TA1537, with or without exogenous metabolic activation from induced hamster or rat liver S9.

During the second study the substance has been tested in 7 different concentrations on S. typhimurium TA 98 and TA 100 and E. coli WP2 uvrA/pKM101 with and without S9 mix. The tested substance showed no mutagenic activity in S. typhimurium strains TA98 or TA100 or in E. coli strain WP2uvrA.

Base on those results, genotoxicity on bacteria for AP 1300 S cannot be considered as conclusive.

IN VITRO CYTOGENICITY STUDY IN MAMMALIAN CELLS OR IN VITRO MICRONUCLEOUS STUDY

No tests on the substance in itself is available, nevertheless one study on the structural analogous Similar Substance 01 (CAS 21850-44-2), testing the Sister Chromatide Exchange has been considered in order to complete the assessment (reported from Cavagnaro J & Cortina TA (1984)[3] in the WHO publication[2]). The test has been conducted on CHO (Chinese hamster ovary cells) exposed to the substance in presence and absence of metabolic activation S-9 mix. During the test no statistically significant increase in the number of exchanges per chromosome or per cell have been observed.

 

IN VITRO GENE MUTATION STUDY IN MAMMALIAN CELLS

In order to assess also the mammalian cell gene mutation potential, data available on AP 1300 S has been taken into account. One study has been conducted on the substance in itself according to the OECD Guideline 476 and to the GLP principles. The substance has been tested in 5 different concentrations on mouse lymphoma L5178Y cells with and without metabolic activation. During the test AP 1300 S does not show any mutagenic effect.

 

IN VIVO SOMATIC CELL GENOTOXICITY STUDY

No tests on the substance in itself are available, nevertheless one study on the structural analogous Similar Substance 01 (CAS 21850-44-2) has been considered in order to complete the assessment. The substance was tested in rats (Sprague-Dawley) by means of unscheduled DNA Synthesis Assay in 5 different doses. No significant increase in the mean nuclear grain count was observed at any dose level compared with the solvent control. No significant increase in the mean nuclear grain count was observed at any dose level compared with the solvent control.

Moreover on the NTP database has been reported a result of an in vivo micronucleus test on mice (B6C3F1). The test has been conducted according to the NTP Standard Protocol and negative results have been observed both in male and in female mice (B6C3F1).

 

The presented studies on the Similar Substances are significant due to the great similarity with AP 1300 S.

Similar Substance 01 (CAS 21850-44-2) has been considered in order to complete the assessment due to the great similarity with AP 1300 S. Similar Substance 01 shares with AP 1300 S the same structure, except for the fact that Similar Substance 01 presents two dibromobutane functional groups linked to the dibromophenol core, instead the dibromo-methylbutane as in the case of AP 1300 S.

As previously mentioned, the Similar Substance 02 (CAS 79-94-7) is a precursor/metabolite of AP 1300 S, therefore the second study has been also considered for the assessment.

The justification for the Read Across approaches has been attached to the Section 13.

[1]Brusick D (1982) Mutagenicity evaluation of 785-104A, 785-104B and 785-104C in the Ames Salmonella/microsome plate test (Final report). Kensington, Maryland, Litton Bionetics, Inc. (Combined Reports Nos. LBI 7655, 7656, and 7657 to Great Lakes Chemical Corporation, West Lafayette, submitted to WHO by the Brominated Flame Retardant Industry Panel).

[2]World Health Organization (WHO, Geneva, 1995); Dr. G.J. van Esch “Tetrabromobisphenol A and Derivatived” (Environmental health criteria; 172)

[3]Cavagnaro J & Cortina TA (1984) In vitrosister chromatid exchange in Chinese hamster ovary cells with GLCC 785-104C (Final report). Vienna, Virginia, Hazleton Biotechnologies Corporation (Report to Great Lakes Chemical Corporation, West Lafayette, submitted to WHO by the Brominated Flame Retardant Industry Panel).


Justification for selection of genetic toxicity endpoint
The test has been conducted according to OECD Guideline, on the substance itself, well documented and in according to the GLP principle.

Justification for classification or non-classification

According to the CLP Regulation (EC n. 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

 

Due to the inconclusive results of the “In vitro Gene Mutation in bacteria”, in order to classify the AP 1300 S for germ cell mutagenicity, it has been considered in a protective manner a positive result.

According to the ECHA Guidance R7.a Table R.7.7-5, in the presence of a positive result in Gene Mutation on bacteria test, a negative result in the “Cytotoxicity in vitro” test and a negative result in “Gene mutation in vivo” test, the substance can be stated as non genotoxic.

In conclusion, the substance is not classified for genetic toxicity according to the CLP Regulation (EC n. 1272/2008).