Reaction mass of Amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro cytogenicity in mammalian cells (OECD473) and in vitro gene mutation in mammalian cells (OECD 490) studies have been conducted with the substance reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide. Read across from dilithium azelate and reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide was used to complete the in vitro gene mutation in bacteria endpoint. 

 

Negative results were produced in two key in vitro studies according to the OECD 471 guideline conducted with read across substances, dilithium azelate and reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide.

 

No indications of mutagenic properties with respect to chromosomal or chromatid damage were observed in the in vitro cytogenicity in mammalian cells study with the test item tested up to the maximum feasible concentration of 500 μg/mL, that led to test item precipitation and/or cytotoxicity in the absence and in the presence of metabolic activation. The test item did not induce mutations or present any chromosomal aberration potential in the in vitro gene mutation assay in mammalian cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: 92/69/EWG, B.14 (Ames-Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix (S9 liver microsomal fraction induced with Phenobarbital and beta-Naphtoflavone)

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 31.6 ... 5000 μg/plate
Concentration range in the main test (without metabolic activation): 31.6 ... 5000 μg/plate

Vehicle / solvent:

- Solvent used: DMSO (Dimethylsulfoxide)

Species / strain:

other: as specified above

Metabolic activation:

with

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 μg/plate)

Species / strain:

other: as specified above

Metabolic activation:

without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 μg/plate)

Additional information on results:

Observations
No toxic effecrs of the test item were observed in both experiments up to the highest investigated concentration in all strains used.

No substantial increases in the revertant colony numbers of any of the five test strains were detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments.

As positive controls reference mutagens were tested in parallel to the test item. They showed a distinct increase of induced revertant colonies. Therefore, the validity and sensitvity of the test system was given.

Remarks on result:

other: Test system other: preliminary test

Conclusions:

In conclusion, the test item was negative with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: 92/69/EWG, B.14 (Ames-Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix (S9 liver microsomal fraction induced with Phenobarbital and beta-Naphtoflavone)

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 31.6 ... 5000 μg/plate
Concentration range in the main test (without metabolic activation): 31.6 ... 5000 μg/plate

Vehicle / solvent:

- Solvent used: DMSO (Dimethylsulfoxide)

Species / strain:

other: as specified above

Metabolic activation:

with

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 μg/plate)

Species / strain:

other: as specified above

Metabolic activation:

without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 μg/plate)

Additional information on results:

Observations
No toxic effecrs of the test item were observed in both experiments up to the highest investigated concentration in all strains used.

No substantial increases in the revertant colony numbers of any of the five test strains were detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments.

As positive controls reference mutagens were tested in parallel to the test item. They showed a distinct increase of induced revertant colonies. Therefore, the validity and sensitvity of the test system was given.

Remarks on result:

other: Test system other: preliminary test

Conclusions:

In conclusion, the test item was negative with and without metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May to 07 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A0296
- Expiration date of the lot/batch: Not reported

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature (+10°C to +25°C), in a tightly closed container, protected from humidity

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was not soluble in water, dimethyl sulfoxide (DMSO) or ethanol. Hence, the test item was dispersed in ethanol to a stable suspension of 50 mg/mL and further diluted to the appropriate lower concentrations.

Species / strain / cell type:

lymphocytes: human peripheral lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human peripheral blood was obtained by venipuncture from young, non-smoking, healthy donor known to be without any medication and collected in heparinised vessels. Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of complete culture medium. The tubes were sealed and incubated at 37°C with occasional shaking to prevent clumping.
- Suitability of cells: The human peripheral lymphocyte is an extremely sensitive indicator of in vitro chromosome damage and can be stimulated to provide large numbers of rapidly dividing cells and metaphases.

For lymphocytes:
- Sex, age and number of blood donors: Blood samples were obtained from young, non-smoking, healthy donors.
- Whether whole blood or separated lymphocytes were used: Whole blood was used.
- Whether blood from different donors were pooled or not: Two replicate cultures were used at each concentration tested, using blood from the
same donor.
- Mitogen used for lymphocytes: None.

MEDIA USED
Complete medium:
100 mL Chromosome Medium with Phytohemagglutinin
1 mL Penicillin/Streptomycin (10 000 IU/mL)

Treatment medium:
500 mL Ham's F-10
13.1 mL Fetal Calf Serum

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Post-mitochondrial fraction (S9 fraction) from male rats treated with Aroclor 1254, prepared according to Maron and Ames (1983) was obtained from Trinova Biochem.
- method of preparation of S9 mix: According to Maron and Ames (1983)
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of S9 mix was added to the appropriate cultures (4.5 mL).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 fraction was examined for the protein content of the S9 fraction and cytochrome P-450 activity by Moltox (Molecular Toxicology, Inc., Boone, NC 28607, USA).

Test concentrations with justification for top dose:

A preliminary study was conducted to establish the top concentration for the main cytogenetic test. In this preliminary experiment concentrations of 1.0, 3.16, 10.0, 31.6, 100, 316 and 500 μg test item/mL medium were employed. Cytotoxicity was noted at concentrations of 316 and 500 μg/mL in the absence of metabolic activation (24-hour exposure). No signs of cytotoxicity were noted in the presence of metabolic activation (4-hour exposure) up to the top concentration of 500 μg/mL medium. Test item precipitation was noted macroscopically starting at a concentration of 100 μg/mL medium in both experiments. No relevant changes in pH or in osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 500 μg/mL medium.
Hence, in the main study the highest concentrations employed were 500 μg/mL medium in the experiments without and with metabolic activation. In the main experiment concentrations of 62.5, 125, 250 and 500 μg test item/mL medium were used.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: The test item was dispersed in ethanol to a stable suspension of 50 mg/mL and further diluted to the appropriate lower concentrations. The vehicle concentration in the medium was 1%.
- Justification for choice of solvent/vehicle: The test item dispersion in ethanol was prepared as the test item was not soluble in water, dimethyl sulfoxide (DMSO) or ethanol. The vehicle ethanol was employed as the negative control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Mitomycin C (0.2 μg/mL) was employed as positive control in the absence of metabolic activation and cyclophosphamide (20 μg/mL) was used as positive control in the presence of metabolic activation.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : Three completely independent experiments were carried out. In experiment 1, the cells were exposed to the test item for 4 hours without S9 mix. In experiment 2, cells were
exposed to the test item for 24 hours without S9 mix, and in experiment 3, the cells were exposed to the test item for 4 hours with S9 mix.

METHOD OF TREATMENT/ EXPOSURE:
- Three independent experiments were carried out. After 48 hours of culture in complete medium the tubes were centrifuged, and the cell pellet was resuspended to 4.5 mL (for S9 mix addition) or 5.0 mL with treatment medium including the test item at the final tested concentrations. The formulations at different concentrations and the controls were added at a volume of 50 μL. S9 mix (0.5 mL) was added to the appropriate cultures (Experiment no. 3). Experiment no. 1 and 2 were carried out without S9 mix. During treatment the tubes were incubated for 4 hours (experiments 1 and 3) or 24 hours (experiment 2) in a shaking water bath at 37°C.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 hours
- Exposure duration/duration of treatment: 4 hours (experiment 1 and 3) or 24 ours exposure (experiment 2)
- Harvest time after the end of treatment (sampling/recovery times): In experiment 1 and 3, during treatment the tubes were incubated for 4 hours. After 4 hours of treatment, the tubes were centrifuged and the cells were washed with 5 mL treatment medium to remove the test item and S9 mix (experiment 3). After further centrifugation the cell pellet was resuspended in 5 mL of complete medium and returned to the incubator for further 20 hours.
In experiment 2, a continuous treatment of 24 hours without metabolic activation was carried out.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Two hours before termination, the cell division was arrested by the addition of 0.5 mL of the spindle poison Colcemid®11 to each culture (10 μg/mL solution). The tubes were capped and incubated for a further two hours. The cells were harvested by low speed centrifugation (80 - 100 x g) and the pellets of cells collected were resuspended in hypotonic potassium chloride solution (0.56%) and fixed in freshly prepared methanol : glacial acetic acid fixative (4:1, v/v).

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The tubes were centrifuged, the fixative removed and the cell pellet resuspended in a few drops of 60% acetic acid. Single drops of the cell suspension were spread on clean, grease-free glass slides on a hot plate (approx. 50°C) and the slides were left to air-dry. Two slides were prepared per culture, stained for 30 minutes in Giemsa12 stain (1:10 in WEISE's buffer12 pH 6.8), washed in buffer and left to air-dry. Permanent slides were made using CONSUL MOUNT13 mountant after clearing in xylene.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): To examine the toxicity of the test item, 1000 cells were scored and the mitotic index was calculated as the percentage of cells in metaphase.

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Observed aberrations were noted and scored according to J. R. K. SAVAGE (1975):
Gap: Achromatic region in chromatid(s) not greater than the width of a chromatid; scored as single-stranded or doublestranded.
Break: Achromatic region in chromatid(s) greater than the width of a chromatid or a discontinuity with displacement; scored as chromatid or chromosomal.
Fragment: Any free displaced portion of chromatid material.
Exchange: Aberration arising from an exchange between two or more chromosomes which results in the products reuniting to form a dicentric or polycentric structure. These may be chromosome or chromatid interchanges. In studies of this type, where full karyotyping is not undertaken and chromosome banding has not been performed, only asymmetrical or chromatid exchanges will normally be recognised.
Other types of aberrations: E.g. isochromatid/isochromatid exchanges such as dicentric chromosomes, centric rings, pulverised metaphases or polyploidy.

In addition, the total number of gaps was recorded for each culture, but excluded from evaluation.
Metaphases which differed from the normal diploid complement (46) were excluded from evaluation. However, test item-related variations of the normal chromosome number were noted (polyploidy / endoreduplication).
Metaphases were assigned to one of the following 5 categories:
1 - normal metaphases
2 - metaphases with 1 - 2 aberrations
3 - metaphases with multiple aberrations
4 - pulverised metaphases:
extreme (≥ 50%) fragmentation of
chromosomal material
5 - polyploidy / endoreduplication
For each treatment and culture 150 metaphases were examined.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: To examine the toxicity of the test item, 1000 cells were scored and the mitotic index was calculated as the percentage of cells in metaphase.

Evaluation criteria:

The test item is considered to have mutagenic properties with respect to chromosomal or chromatid change, if the following criteria are fulfilled:
o The number of chromosomal aberrations is significantly (at p ≤ 0.05) increased compared with the solvent control in at least one of the test concentrations
o The increase observed is concentration-dependent
o The increase should not occur in the severely cytotoxic range (mitotic index <0.25), as it is known that high cytotoxicity causes artefacts in the form of aberrations in in vitro chromosomal aberration tests
o Any of the results are outside the distribution of the historical negative control data
o A reproducible increase in the number of cells with chromosomal aberrations
o All three experimental conditions were tested unless one resulted in positive results

Acceptance of the test was based on the following criteria:
o The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
o Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
o All three experimental conditions were tested unless one resulted in positive results (see paragraph 28 of the OECD 473).
o Adequate number of cells and concentrations are analysable (paragraphs 31 and 21 of the OECD 473).
o The criteria for the selection of top concentration are consistent with those described in paragraphs 22, 23 and 24 of the OECD 473.
o Both replicate cultures lead to similar results.
o Adequate numbers of cells (i.e. at least 1000 countable cells) and concentrations re-analyzable.

Statistics:

The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R. A. FISHER (p ≤ 0.05).

Species / strain:

other: human peripheral lymphocyte

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

No cytotoxicity in the presence of metabolic activation but cytotoxicity was recorded in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH of the medium, negative control and the test item formulations in the medium were determined prior to the main test. The pH values were in the range of 7.54 and 7.68.
- Data on osmolality: Osmolarity of the medium, negative control and the test item formulations in the medium was measured using a semi-micro osmometer (type ML) prior to the main test. The osmolarity values were in the range of 280 and 455 mOsmol/kg.
- Water solubility: The solubility was assessed at the beginning and the end of treatment, as solubility could change during the course of exposure in the test system due to presence of cells, S9, serum etc. Insolubility would be detected by using the unaided eye. The precipitate should not interfere with the scoring. The test item was not soluble in water, dimethyl sulfoxide (DMSO) or ethanol. Hence, the test item was dispersed in ethanol to a stable suspension of 50 mg/mL and further diluted to the appropriate lower concentrations. Hence, the maximum feasible final concentration in the medium was 500 μg/mL medium employing a vehicle (ethanol) at concentration of 1%.
- Precipitation and time of the determination: Test item precipitation was noted macroscopically starting at a concentration of 100 μg/mL.

RANGE-FINDING/SCREENING STUDIES: Two preliminary experiments were conducted: 1) without S9 mix (24-hour exposure) and 2) in the presence of S9 mix (4-hour exposure). Cytotoxicity was noted at concentrations of 316 and 500 μg/mL in the absence of metabolic activation (24-hour exposure). No signs of cytotoxicity were noted in the presence of metabolic activation (4-hour exposure) up to the top concentration of 500 μg/mL. Test item precipitation was noted macroscopically starting at a concentration of 100 μg/mL medium in both experiments. No relevant changes in pH or in osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 500 μg/mL medium

STUDY RESULTS :
- Tests without metabolic activation (4- and 24-hour exposure): The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with test item at concentrations ranging from 62.5 to 500 μg/mL in the absence of metabolic activation ranged from 0.0% to 1.0%. These results are within the range of the historical control data (0% to 3%).
- Test with metabolic activation (4-hour exposure): The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with test item at concentrations ranging from 62.5 to 500 μg/mL in the presence of metabolic activation ranged from 0.0% to 1.7%. These results were within the range of the historical control data (0% to 2.7%).

- Concurrent vehicle negative and positive control data :
Test without metabolic activation (4- and 24-hour exposure): The vehicle control cultures had a mean of cells with aberrations (excluding gaps) of 0.0% (4-hour or 24-hour exposure). The positive control cultures had a significantly increased mean frequency of cells with aberrations (excluding gaps) of 8.7% or 9.7% (4-hour or 24-hour exposure, respectively), which was in line with the historical control range. Therefore, the test is considered to be valid.
Test with metabolic activation (4-hour exposure): The vehicle control cultures had a mean of 1.0% cells with aberrations (excluding gaps), and the positive control cultures had a significantly increased mean frequency of cells with aberrations of 9.3%, which was in line with the historical control range. Therefore, the test is considered to be valid.

Table 1. Results of the preliminary cytotoxicity test 

Concentration of test item (μg/mL)	S9 mix	Metaphases/1000 cells	Mitotic index#1
	4-hour exposure
0	+	72	1.00
1	+	52	0.72
3.16	+	62	0.86
10	+	62	0.86
31.6	+	56	0.78
100*	+	58	0.81
316*	+	58	0.81
500*	+	68	0.94
	24-hour exposure
0	-	36	1.00
1	-	26	0.72
3.16	-	35	0.97
10	-	35	0.97
31.6	-	29	0.81
100*	-	31	0.86
316*	-	16	0.44
500*	-	16	0.44

*Test item precipitation

#1 = mitotic index: number of metaphases/1000 cells;

+ = with metabolic activation

- = without metabolic activation

 

Table 2. Summary of results of the experiments without metabolic activation (S9 mix)

 

Treatment (μg test item/mL)	Mitotic index#1	Number of metaphases scored	% of cells with aberrations excluding gaps	Mitotic index#1	Number of metaphases scored	% of cells with aberrations excluding gaps
	4-hour exposure			24-hour exposure
Vehicle control (1 % ethanol)	1.00	300	0.0	1.00	300	0.0
62.5	0.81	300	1.0	1.14	300	0.3
125*	1.02	300	0.3	1.11	300	0.3
250*	0.93	300	0.0	0.64	300	1.0
500*	0.76	300	0.3	0.31	83#2	0.0
Positive control (0.2 µg mitomycin /mL)	1.11	300	8.7s	0.23	300	9.7s

s. = significantly different from negative control (p < 0.05)

* = test item precipitation

#1 = mitotic index: number of metaphases/1000 cells; negative control = 1.00

#2 = no more metaphases of sufficient quality for evaluation due to cytotoxicity of the test item

 

Table 3.Summary of results of the experiments with metabolic activation (S9 mix)

Treatment (μg test item/mL)	Mitotic index#1	Number of metaphases scored	% of cells with aberrations excluding gaps
	4-hour exposure
Vehicle control (1 % ethanol)	1.00	300	1.0
62.5	1.36	300	0.0
125*	1.23	300	1.7
250*	1.65	300	0.0
500*	1.00	300	1.3
Positive control (20 µg cyclophosphamide/mL)	0.58	300	9.3s

s. = significantly different from negative control (p < 0.05)

* = test item precipitation

#1 = mitotic index: number of metaphases/1000 cells; negative control = 1.00

Conclusions:

Under the test conditions, the test item tested up to the maximum feasible concentration of 500 μg/mL, that led to test item precipitation and/or cytotoxicity in the absence and in the presence of metabolic activation, revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.

Executive summary:

The potential of the test item to induce chromosomal aberrations was examined in an in vitro cytogenetic study according to OECD 473 guideline. The study was conducted using human lymphocyte cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. The main test was conducted in three independent experiments with four test concentrations of 62.5, 125, 250 and 500 μg test item/mL. In experiment 1, the cells were exposed to the test item for 4 hours without S9 mix. In experiment 2, cells were exposed to the test item for 24 hours without S9 mix, and in experiment 3, the cells were exposed to the test item for 4 hours with S9 mix. The harvesting time was 24 hours after initiation of exposure. Two replicate cultures were used at each concentration tested. Mitomycin C (0.2 μg/mL) was employed as positive control in the absence of metabolic activation and cyclophosphamide (20 μg/mL) was employed as positive control in the presence of metabolic activation.

No signs of cytotoxicity were noted with and without metabolic activation up to the top concentration of 500 μg/mL following 4-hour exposure. In the 24-hour exposure experiment without metabolic activation cytotoxicity was noted at the top concentration of 500 μg/mL. Test item precipitation was noted macroscopically starting at a concentration of 125 μg/mL in all experiments. Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.

The study is GLP-compliant, guideline study, and has been assigned a Klimisch score of 1.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May 2018 to 26 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted 29 July, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

dated 30 May, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A0296
- Expiration date of the lot/batch: Not reported

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature (+10°C to +25°C), in a tightly closed container, protected from humidity

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test material was not soluble in water, dimethyl sulfoxide (DMSO) or ethanol. Hence, the test item was dispersed in ethanol to a stable suspension of 50 mg/mL and further diluted to the appropriate lower concentrations.
- Final dilution of a dissolved solid: The maximum feasible final concentration in the medium was 500 μg/mL employing a vehicle concentration of 1%.

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: The pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment employing the methods given below:
pH values: using a digital pH meter (SevenCompact™ pH/Ion S210)
Osmolality: with a semi-micro osmometer
No changes in pH of the test item formulations at concentrations up to 500 μg/mL compared to the negative control were noted that could affect the validity of the assay. Further, no changes in osmolality of the test item formulations at concentrations up to 250 μg/mL compared to the negative control were noted that could affect the validity of the assay. The osmolality at the top concentration of 500 μg/mL was increased to 620 mOsmol/kg but did not influence the mutation frequency and, hence, did not affect the validity of this experiment.

Target gene:

thymidine kinase (TK) locus in L5178Y TK +/- mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y mouse lymphoma cell line which is heterozygotous at the TK locus (+/-). The cell line was isolated by ATCC and stock culutres were obtained from ATCC (American Type Culture Collection, 0801 University Blvd., Manassas, VA 20110-2209, USA).
- Suitability of cells: Thymidine kinase (TK) is a cellular enzyme that allows cells to salvage thymidine from the surrounding medium for use in DNA synthesis. If the thymidine analogue 5-trifluorothymidine (TFT) is included in the growth medium, the analogue will be phosphorylated via the TK pathway and will cause cell death by inhibiting DNA synthesis. Cells, which are heterozygous at the TK locus (TK +/-), may undergo a single-step forward mutation
to the TK-/- genotype in which little or no TK activity remains. Such mutants are as viable as the heterozygotes in normal medium because DNA synthesis may still proceed by de novo synthetic pathways that do not involve thymidine as an intermediate. The basis for selection of the TK-/- mutants is the lack of any ability to utilise toxic analogues of thymidine, which enable only the TK-/-mutants to grow in the presence of TFT. Cells, which grow to form colonies in the presence of TFT, are therefore assumed to have mutated, either spontaneously or induced by the test item, to the TK-/- genotype.

For cell lines:
- Absence of Mycoplasma contamination: Laboratory cultures were periodically checked for the absence of mycoplasma contamination by culturing methods.
- Methods for maintenance in cell culture: Stock cultures were obtained from ATCC, and master stocks were maintained in liquid nitrogen.
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: The cells used during the experimental studies were maintained in growth medium RPMI 16401 with glutamax medium supplemented with Pluronic® F68, gentamycin, amphotericin B and horse serum (10% by volume). Treatment medium is growth medium with a reduced horse serum content (5% by volume). Plating medium is growth medium with increased horse serum content (approx. 20% by volume) but without Pluronic® F68. Selection medium is growth medium that contains 3 μg/mL of TFT3.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : S9 was collected from male rats treated with Aroclor 1254
to induce mixed function oxidase enzymes capable of transforming chemicals to more active forms.
- method of preparation of S9 mix: The post-mitochondrial fraction (S9 fraction) prepared according to MARON and AMES (1983) was obtained from Trinova Biochem. The S9 fraction was stored at below -80°C until use. The S9 fraction was thawed immediately before use and was combined to form the activation system. The medium/S9 mix was sterilised through a 0.45 μm filter and kept on ice at all times.
- volume of S9 mix and S9 in the final culture medium: 3 mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes, quality of S9 mix was confirmed (Certificate attached to the study report).

Test concentrations with justification for top dose:

Preliminary study: 3.16, 10.0, 31.6, 100, 316 and 500 μg/mL (without and with metabolic activation).
Main study: 31.25, 62.5, 125, 250 and 500 μg/mL (without and with metabolic activation).

In the preliminary test, cytotoxicity (decreased survival and a relative plating efficiency (RPE) of 21%) was noted at the top
concentration of 500 μg/mL in the absence of metabolic activation (24-hour exposure). A slightly decreased viability (decreased survival and a relative plating efficiency (RPE) of 53%) was noted at the top concentration of 500 μg/mL in the presence of metabolic
activation (3-hour exposure). Based on the results of preliminary study, the highest concentrations employed in the main study were 500 μg/mL in the experiments without and with metabolic activation. The separation factor of 2 was used. No increase in the mutant frequency was observed. Hence, it was considered acceptable not to add any further lower concentrations, as these additional lower concentrations would provide no further information.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

- Justification for choice of solvent/vehicle: Test item was not soluble in water, dimethyl sulfoxide (DMSO) or ethanol. Hence, the test item was dispersed in ethanol to a stable suspension of 50 mg/mL and further diluted to the appropriate lower concentrations.

- Justification for percentage of solvent in the final culture medium: The maximum feasible final concentration in the medium was 500 μg/mL employing a vehicle concentration of 1%.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Remarks:

Positive controls were not used in the cytotoxicity assay.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours, the experiment with S9 mix was carried out in two independent assays.
- Number of cultures per concentration: single
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
The cells for the first and second experiments were obtained from logarithmically growing laboratory stock cultures. The cells were pelleted by centrifugation, the culture medium was removed, and the cells were re-suspended in treatment medium that contained 5% heat inactivated horse serum, and the corresponding concentration of test substance was added. The final cell density at start of treatment was 0.5 x 10E6
cells/mL. The dosed tubes were closed, mixed, and placed on a roller drum at approx. 37°C at approx. 40 rpm for an exposure period of 3 hours.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: An exposure time of 3 hours was used in assays with and without metabolic activation. The non-activation and activation experiments were repeated in an independent
experiments. An exposure time of 3 hours was used for the repeat experiment with metabolic activation and an exposure time of 24 hours instead of 3 hours was used for the repeat experiment without metabolic activation covering long term effects.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): After the exposure, the cells were washed and re-suspended in growth medium. Cell densities were adjusted to 2 x 10E5 cells/mL and incubated for 2 days for the expression period. Within the expression period cell densities were determined after incubation for 24 hours and, after adjustment to 2 x 10E5 cells/mL, the cells were incubated for another 24 hours.
- Method used: For the plating for survival, one aliquot of each cell suspension was diluted to 8 cells/mL in plating medium and 0.2 mL of each culture were placed in each well of two 96 well microtiter plates (^ 192 wells, average 1.6 cells/well). After 1-week incubation, the number of viable clones was recorded.
For the TFT resistance TFT was added to the cultures (final concentration 3 μg/mL) and 0.2 mL of each cell suspension were placed into each well of four 96-well microtiter plates (^ 384 wells, average 2 x 10E3 cells/well). The plates were incubated for 10 to 12 days and wells containing clones were identified microscopically and counted.
- Criteria for small (slow growing) and large (fast growing) colonies: Large colonies are defined as ≥ 1/4 and small colonies < 1/4 of the well diameter of 6 mm.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cytotoxicity was defined as the Relative Total Growth (RTG) which includes the Relative Suspension Growth (RSG) during the 2 day expression period and the Relative Plating Efficiency (RPE) obtained at the time of mutant selection. RTG, RSG and RPE were all expressed as a percentage and were calculated as follows:
RTG = RSG x RPE

RSG = [SG value (treatment)/Mean SG value (control)] x 100
With SG being the suspension growth, the measure of the growth in suspension during treatment and the expression period. SG was calculated as follows:
SG = a x b x c
where:
a = D0 post-treatment cell count/Pre-treatment cell density,
b = D1 cell count/Cell count set up on D0 post-treatment,
c = D2 cell count/Cell count set up on D1

RPE = [PE value (treatment)/Mean PE value (control)] x 100

With PE being the plating efficiency obtained at the time of mutant selection. The calculations are based on P, the probable number of clones per well:
PE = P / Number of cells plated per well
where: P = -ln [Number of wells with no colony/Total number of wells]

The cloning efficiency (CE) is defined as the plating efficiency in percent (CE = PE x 100).

- Any supplementary information relevant to cytotoxicity: For the evaluation of the cytotoxicity in the preliminary experiment the relative plating efficiency RPE obtained at the time after treatment is sufficient and was evaluated. The RPE was calculated as described above for the main experiment.

METHODS FOR MEASUREMENTS OF GENOTOXICIY :

- Results of determination of mutant frequency:
The mutant frequency is expressed as mutants/10E6 viable cells as follows:
Mutant frequency (MF/10E6) = [PEM of mutant cells/PE of viable cells] x 10E6

- Results of the determination of large and small colonies, i.e. number of wells containing large colonies and number of wells containing small colonies in the negative control, the positive controls, and the different test item concentrations.

- Analysis of mutant colony size: The percentage of large colonies and of small colonies, and the small : large ratio were determined.

Evaluation criteria:

Evaluation criteria are based on recommendations of The Mouse Lymphoma Expert Workgroup (Moore et al. 2006).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No changes in pH of the test item formulations at concentrations up to 500 μg/mL compared to the negative control were noted that could affect the validity of the assay.
- Data on osmolality: No changes in osmolality of the test item formulations at concentrations up to 250 μg/mL compared to the negative control were noted that could affect the validity of the assay. The osmolality at the top concentration of 500 μg/mL was increased to 620 mOsmol/kg but did not influence the mutation frequency and, hence, did not affect the validity of this experiment.
- Possibility of evaporation from medium: None reported.
- Water solubility: Test item was not soluble in water.
- Precipitation: In the preliminary test, precipitation was observed at test concentration of 100 μg/mL and above. In the main test, precipitation was observed at test concentration of 125 μg/mL and above,

RANGE-FINDING/SCREENING STUDIES (if applicable): Cytotoxicity (decreased survival and a relative plating efficiency (RPE) of 21%) was noted at the top concentration of 500 μg/mL in the absence of metabolic activation (24-hour exposure).
No signs of cytotoxicity were noted in the presence of metabolic activation (3-hour exposure). A slightly decreased viability (decreased survival and a relative plating efficiency (RPE) of 53%) was noted at the top concentration of 500 μg/mL in the presence of metabolic activation (3-hour exposure).

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency of 330.29 and 586.71 mutant colonies per 106 cells in the case of MMS and of 694.62 and 800.00 mutant colonies per 106 cells in the case of 3-MC. All positive controls showed an increase in the small colony MF of at least
150 x 10-6 above that seen in the concurrent solvent control and an absolute increase in total mutation frequency of at least 300 x 10-6. Furthermore, the mean relative total growth (RTG) for the positive controls was greater than or equal to 10%. Hence, the acceptance criteria were met.
The negative controls had mutation frequencies of 53.56 or 52.02 mutant colonies per 10E6 cells in the experiments without metabolic activation (3- or 24-hour exposure, respectively) and 56.71 or 51.73 mutant colonies per 10E6 cells in the experiments with metabolic activation, and, were all well within the historical data-range. The calculations of suspension growth (SG) of the negative controls were in the acceptance criteria range between 8 and 32 following 3-hour treatments or between 32 and 180 following 24-hour treatments. The mean cloning efficiencies (CE = PE x 100) of the negative controls from the Mutation Experiments were between the range 65% to 120%. Hence, the acceptance criteria were met.
The mutation frequency and the colony size ratio of the positive controls and negative controls without and with metabolic activation for the experiments of the years 2015 to 2017 (background data).

For all test methods and criteria for data analysis and interpretation:
To define positive and negative results and to assure that the increased MF is biologically relevant instead of a statistical analysis (generally used for other tests), the interpretation relies on the use of a predefined induced mutant frequency (i.e. increase in MF above concurrent control), designated as the Global Evaluation Factor (GEF). The GEF (126 x 10E-6) is based on the analysis of the distribution of the negative control MF data (Moore et al., 2006).

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
Decreased relative total growth (RTG) was noted at a concentration of 500 μg/mL in the absence (24-hour exposure) and presence (3-hour exposure) of metabolic activation and, in addition, at 250 μg/mL in the 24-hour exposure experiment. The results are attached as a background material (Table 2a and 2b).

- Genotoxicity results:
In the experiments without metabolic activation, the mutation frequencies of the cultures treated with test item ranged from 49.01 to 86.03 mutant colonies per 10E6 cells (3 hours exposure) and from 50.07 to 91.06 mutant colonies per 10E6 cells (24 hours exposure). In the experiments with metabolic activation, mutation frequencies ranged from 54.61 to 89.82 mutant colonies per 10E6 cells (3 hours exposure, first assay) and from 53.27 to 114.77 mutant colonies per 10E6 cells (3 hours exposure, second assay). The results are attached as a background material (Table 3a and 3b). No change was observed in the ratio of small to large mutant colonies, ranging from 0.21 to 1.50 for test item-treated cells and ratios of 0.65 to 1.00 for the negative controls. The results are attached as a background material (Table 4a and 4b).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Positive historical control data are available from 2015 to 2017 (n = 19), the results are attached as a background material.
- Negative (solvent/vehicle) historical control data: Vehicle historical control data are available from 2015 to 2017 (n = 19), the results are attached as a background material.

Table 1. pH and osmolality data of the negative control and of all test item formulations in the medium determined in the preliminary test:

 

 

Concentration [μg test item/mL medium]	pH value	Osmolality [mOsmol/kg]
Medium	7.44	280
Negative control	7.30	455
3.16	7.32	450
10	7.32	450
31.6	7.34	450
100	7.32	450
316	7.39	450
500	7.37	620

 

Conclusions:

Under the test conditions, the test item, tested up to the maximum feasible concentration of 500 μg/mL, that led to test item precipitation and/or cytotoxicity, neither induced mutations nor had any chromosomal aberration potential.

Executive summary:

The potential of the test item to induce gene mutations was examined in an in vitro study according to OECD 490 guideline. The study was conducted using cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours (the experiment with S9 mix was carried out in two independent assays). The test item was not soluble in water, dimethyl sulfoxide (DMSO) or ethanol, therefore was dispersed in ethanol to a stable suspension of 50 mg/mL and further diluted to the appropriate lower concentrations. A preliminary study was conducted to establish the highest concentration for the main study. The main study was conducted with five test concentrations of 31.25, 62.5, 125, 250 and 500 μg test item/mL.

Methylmethanesulfonate (15 or 12 μg/mL for a 3- and 24-hour exposure, respectively) was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (1.0 μg/mL) in the presence of exogenous metabolic activation. The vehicle ethanol was employed as the negative control.

 

Under the test conditions, test item tested up to the maximum feasible concentration of 500 μg/mL, that led to test item precipitation and/or cytotoxicity, in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. The positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.

No change was noted in the ratio of small to large mutant colonies. Therefore, test item did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that the test item, tested up to the maximum feasible concentration of 500 μg/mL, that led to test item precipitation and/or cytotoxicity, neither induced mutations nor had any chromosomal aberration potential.

The study is GLP-compliant, guideline study, and has been assigned a Klimisch score of 1.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as an unpublished report.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

Each S9 batch is characterized with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively Not the correct amount of 2-aminoanthracene was mentioned in the protocol. This writing error in the protocol had no effect on the results of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Salmonella: +Histidine
- E.Coli: Tryptophan

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9-mix

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment one: 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment two: 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Milli Q water for test substance and DMSO for positive controls (except sodium azelate which used saline)
- Preparation: Test substance concentrations were used within 3 hours after preparation.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2.5 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2.5 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate in direct plate assay and 5 µg/plate in preincubation assay

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 15 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Sodium azide: 5 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR 191: 2.5 µg/plate and 2-nitrofluorene: 10 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene: 10 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Methylmethanesulfonate: 650 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline N-oxide: 10 µg/plate

Remarks:

Without S9 mix

Details on test system and experimental conditions:

METHODS OF APPLICATION
- Experiment 1: In agar (plate incorporation)
- Experiment 2: Pre-incubation

DURATION
- Preincubation period for bacterial strains: 30 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
To determine the toxicity, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Evaluation criteria:

Acceptability of the assay
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Data evaluation and statistical procedures
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done. Standard deviation was determined.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results: The substance was initially tested in the tester strains TA100 and WP2uvrA as a dose range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate. In the absence of S9-mix in tester strain TA1535, the mean plate count of the solvent control (3) was below the limit of the range (6). Therefore this part was repeated in an additional (second experiment), the dose range tested was 52, 164, 512, 1600 and 5000 μg/plate in the absence of S9-mix.
Precipitate: Precipitation of the substance on the plates was not observed at the start or at the end of the incubation period in all tester strains.
Toxicity: In the direct plate assay, no reduction of the bacterial background lawn was observed. No decrease in the number of revertants less than the minimal value of the historical control data range were observed in all tester strains tested. However a high solvent control value in tester strain TA1537 in the absence of S9-mix resulted in a doubtful toxic observation. Therefore this part of the experiment was repeated in an additional (second experiment), the dose range tested was 52, 164, 512, 1600 and 5000 μg/plate in the absence of S9-mix. In the pre-incubation assay, there was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Mutagenicity: In the direct plate test and the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
Negative controls: The negative control values were within the laboratory historical control data ranges.
Positive controls: The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Dose range finding test - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Mean number of revertant colonies/3 replicate plates (±S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain

	Without S9		With S9
Dose	TA100	WP2uvrA	TA100	WP2uvrA
Positive control	798 ± 110	1375 ± 91	1312 ± 105	217 ± 7
Solvent control	104 ± 11	21 ± 5	107 ± 18	25 ± 7
1.7	96 ± 6	33 ± 6	111 ± 5	27 ± 4
5.4	90 ± 15	23 ± 6	102 ± 15	29 ± 16
17	100 ± 3	26 ± 6	95 ± 9	31 ± 13
52	104 ± 5	21 ± 6	124 ± 7	34 ± 8
164	91 ± 7	21 ± 3	108 ± 6	39 ± 5
512	99 ± 17	25 ± 8	97 ± 9	22 ± 10
1600	99 ± 3	24 ± 6	97 ± 3	33 ± 7
5000	99 ± 6 *	27 ± 4 *	113 ± 23 *	30 ± 6 *

* No precipitate and normal bacterial background lawn

Table 2: Experiment 1 - Mutagenic responnse in Salmonella typhimurium reverse mutation assay - Direct plate assay

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium

	Without S9			With S9
Dose	TA 1535	TA 1537	TA 98	TA 1535	TA 1537	TA 98
Positive control	750 ± 26	868 ± 40	1052 ± 46	209 ± 10	337 ± 37	943 ± 40
Solvent control	3 ± 2	15 ± 6	16 ± 5	9 ± 3	8 ± 5	23 ± 2
52	10 ± 3	3 ± 3	18 ± 6	8 ± 3	10 ± 2	24 ±8
164	10 ± 6	4 ± 2	17 ± 5	11 ± 4	8 ± 4	14 ± 3
512	13 ± 3	4 ± 4	18 ± 4	11 ± 2	6 ± 2	20 ± 8
1600	18 ± 2	5 ± 2	14 ± 5	14 ± 6	11 ± 3	22 ± 8
5000	13 ± 4 *	9 ± 3 *	14 ± 7 *	11 ± 4 *	4 ± 1 *	23 ± 6 *

* No precipitate and normal bacterial background lawn

Table 3: Experiment 2 - Mutagenic response in the Salmonella typhimurium reverse mutation assay - Direct plate assay

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium

	Without S9
Dose	TA100	WP2uvrA
Positive control	773 ± 22	667 ± 58
Solvent control	13 ± 6	6 ± 4
52	11 ± 10	5 ± 4
164	16 ± 3	8 ± 4
512	21 ± 7	5 ± 2
1600	10 ± 3	6 ± 4
5000	14 ± 2 *	3 ± 4 *

* No precipitate and normal bacterial background lawn

Table 4: Experiment 2 - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay - Pre-incubation assay

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

	Without S9					With S9
Dose	TA 1535	TA 1537	TA 98	TA 100	WP2uvra	TA 1535	TA 1537	TA 98	TA 100	WP2uvra
Positive control	791 ± 18	63 ± 14	974 ± 26	697 ± 91	158 ± 74	134 ± 12	169 ± 13	512 ± 17	2137 ± 204	343 ± 9
Solvent control	15 ± 1	4 ± 1	20 2) 3	97 ± 15	28 ± 9	10 ± 2	4 ± 3	35 ± 2	96 2) 16	30 ± 7
52	20 ± 6	3 ± 2	15 ± 4	106 ± 5	24 ± 4	12 ± 4	7 ± 4	27 ± 8	108 ± 14	42 ± 12
164	19 ± 7	6 ± 2	20 ± 6	87 ± 10	21 ± 8	19 2) 5	10 ± 2	28 ± 8	100 ± 15	39 ± 5
512	13 ± 4	8 ± 4	16 ± 10	102 ± 8	20 ± 6	11 ± 4	8 ± 4	26 ± 7	89 ] 11	31 ± 5
1600	15 ± 3	4 ± 3	16 ± 4	91 ± 14	24 ± 5	18 ± 3	8 ± 4	28 ± 6	80 ± 9	31 ± 3
5000	13 ± 3 *	5 ± 2 *	13 ± 3	109 ± 12 *	22 ± 3 *	11 ± 1 *	9 ± 3 *	19 ± 10 *	88 ± 9 *	29 ± 6 *

* No precipitate and normal bacterial background lawn

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study, it is concluded that dilithium azelate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The in vitro mutagenicity of dilithium azelate was assessed in a GLP-compliant Bacterial Reverse Mutation Test, following OECD guideline 471 (WIL 2015). S. typhimurium and E. coli strains were treated with suspensions of dilithium azelate using both the Ames plate incorporation and pre-incubation methods at five dose levels in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The vehicle and positive controls confirmed the sensitivity of the assay and the efficacy of the S9 -mix.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as an unpublished report.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

Each S9 batch is characterized with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively Not the correct amount of 2-aminoanthracene was mentioned in the protocol. This writing error in the protocol had no effect on the results of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Salmonella: +Histidine
- E.Coli: Tryptophan

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9-mix

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment one: 52, 164, 512, 1600 and 5000 µg/plate
- Main test experiment two: 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Milli Q water for test substance and DMSO for positive controls (except sodium azelate which used saline)
- Preparation: Test substance concentrations were used within 3 hours after preparation.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2.5 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2.5 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate in direct plate assay and 5 µg/plate in preincubation assay

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 15 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Sodium azide: 5 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR 191: 2.5 µg/plate and 2-nitrofluorene: 10 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene: 10 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Methylmethanesulfonate: 650 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Milli Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline N-oxide: 10 µg/plate

Remarks:

Without S9 mix

Details on test system and experimental conditions:

METHODS OF APPLICATION
- Experiment 1: In agar (plate incorporation)
- Experiment 2: Pre-incubation

DURATION
- Preincubation period for bacterial strains: 30 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
To determine the toxicity, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Evaluation criteria:

Acceptability of the assay
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Data evaluation and statistical procedures
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done. Standard deviation was determined.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results: The substance was initially tested in the tester strains TA100 and WP2uvrA as a dose range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate. In the absence of S9-mix in tester strain TA1535, the mean plate count of the solvent control (3) was below the limit of the range (6). Therefore this part was repeated in an additional (second experiment), the dose range tested was 52, 164, 512, 1600 and 5000 μg/plate in the absence of S9-mix.
Precipitate: Precipitation of the substance on the plates was not observed at the start or at the end of the incubation period in all tester strains.
Toxicity: In the direct plate assay, no reduction of the bacterial background lawn was observed. No decrease in the number of revertants less than the minimal value of the historical control data range were observed in all tester strains tested. However a high solvent control value in tester strain TA1537 in the absence of S9-mix resulted in a doubtful toxic observation. Therefore this part of the experiment was repeated in an additional (second experiment), the dose range tested was 52, 164, 512, 1600 and 5000 μg/plate in the absence of S9-mix. In the pre-incubation assay, there was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Mutagenicity: In the direct plate test and the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
Negative controls: The negative control values were within the laboratory historical control data ranges.
Positive controls: The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Dose range finding test - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Mean number of revertant colonies/3 replicate plates (±S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain

	Without S9		With S9
Dose	TA100	WP2uvrA	TA100	WP2uvrA
Positive control	798 ± 110	1375 ± 91	1312 ± 105	217 ± 7
Solvent control	104 ± 11	21 ± 5	107 ± 18	25 ± 7
1.7	96 ± 6	33 ± 6	111 ± 5	27 ± 4
5.4	90 ± 15	23 ± 6	102 ± 15	29 ± 16
17	100 ± 3	26 ± 6	95 ± 9	31 ± 13
52	104 ± 5	21 ± 6	124 ± 7	34 ± 8
164	91 ± 7	21 ± 3	108 ± 6	39 ± 5
512	99 ± 17	25 ± 8	97 ± 9	22 ± 10
1600	99 ± 3	24 ± 6	97 ± 3	33 ± 7
5000	99 ± 6 *	27 ± 4 *	113 ± 23 *	30 ± 6 *

* No precipitate and normal bacterial background lawn

Table 2: Experiment 1 - Mutagenic responnse in Salmonella typhimurium reverse mutation assay - Direct plate assay

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium

	Without S9			With S9
Dose	TA 1535	TA 1537	TA 98	TA 1535	TA 1537	TA 98
Positive control	750 ± 26	868 ± 40	1052 ± 46	209 ± 10	337 ± 37	943 ± 40
Solvent control	3 ± 2	15 ± 6	16 ± 5	9 ± 3	8 ± 5	23 ± 2
52	10 ± 3	3 ± 3	18 ± 6	8 ± 3	10 ± 2	24 ±8
164	10 ± 6	4 ± 2	17 ± 5	11 ± 4	8 ± 4	14 ± 3
512	13 ± 3	4 ± 4	18 ± 4	11 ± 2	6 ± 2	20 ± 8
1600	18 ± 2	5 ± 2	14 ± 5	14 ± 6	11 ± 3	22 ± 8
5000	13 ± 4 *	9 ± 3 *	14 ± 7 *	11 ± 4 *	4 ± 1 *	23 ± 6 *

* No precipitate and normal bacterial background lawn

Table 3: Experiment 2 - Mutagenic response in the Salmonella typhimurium reverse mutation assay - Direct plate assay

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium

	Without S9
Dose	TA100	WP2uvrA
Positive control	773 ± 22	667 ± 58
Solvent control	13 ± 6	6 ± 4
52	11 ± 10	5 ± 4
164	16 ± 3	8 ± 4
512	21 ± 7	5 ± 2
1600	10 ± 3	6 ± 4
5000	14 ± 2 *	3 ± 4 *

* No precipitate and normal bacterial background lawn

Table 4: Experiment 2 - Mutagenic response in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay - Pre-incubation assay

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

	Without S9					With S9
Dose	TA 1535	TA 1537	TA 98	TA 100	WP2uvra	TA 1535	TA 1537	TA 98	TA 100	WP2uvra
Positive control	791 ± 18	63 ± 14	974 ± 26	697 ± 91	158 ± 74	134 ± 12	169 ± 13	512 ± 17	2137 ± 204	343 ± 9
Solvent control	15 ± 1	4 ± 1	20 2) 3	97 ± 15	28 ± 9	10 ± 2	4 ± 3	35 ± 2	96 2) 16	30 ± 7
52	20 ± 6	3 ± 2	15 ± 4	106 ± 5	24 ± 4	12 ± 4	7 ± 4	27 ± 8	108 ± 14	42 ± 12
164	19 ± 7	6 ± 2	20 ± 6	87 ± 10	21 ± 8	19 2) 5	10 ± 2	28 ± 8	100 ± 15	39 ± 5
512	13 ± 4	8 ± 4	16 ± 10	102 ± 8	20 ± 6	11 ± 4	8 ± 4	26 ± 7	89 ] 11	31 ± 5
1600	15 ± 3	4 ± 3	16 ± 4	91 ± 14	24 ± 5	18 ± 3	8 ± 4	28 ± 6	80 ± 9	31 ± 3
5000	13 ± 3 *	5 ± 2 *	13 ± 3	109 ± 12 *	22 ± 3 *	11 ± 1 *	9 ± 3 *	19 ± 10 *	88 ± 9 *	29 ± 6 *

* No precipitate and normal bacterial background lawn

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study, it is concluded that dilithium azelate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The in vitro mutagenicity of dilithium azelate was assessed in a GLP-compliant Bacterial Reverse Mutation Test, following OECD guideline 471 (WIL 2015). S. typhimurium and E. coli strains were treated with suspensions of dilithium azelate using both the Ames plate incorporation and pre-incubation methods at five dose levels in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The vehicle and positive controls confirmed the sensitivity of the assay and the efficacy of the S9 -mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The In vitro cytogenicity in mammalian cells and in vitro gene mutation in mammalian cells studies have been conducted withthe substance reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide. Read across from dilithium azelate and reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide was used to complete the in vitro gene mutation in bacteria endpoint.

 

The in vitro mutagenicity of dilithium azelate was assessed in a GLP-compliant Bacterial Reverse Mutation Test, following OECD guideline 471 (WIL 2015). Strains of S. typhimurium and E. coli were treated with suspensions of dilithium azelate using both the Ames plate incorporation and pre-incubation methods at five dose levels in triplicate, both with and without the addition of a rat liver homogenate metabolising system. Based on the results of this study, it is concluded that dilithium azelate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. The study is GLP-compliant, guideline study, and has been assigned a Klimisch score of 1.

The in vitro mutagenicity of reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide was assessed in a GLP-compliant Bacterial Reverse Mutation Test, following OECD guideline 471 (BIOSERVICE SCIENTIFIC LABORATORIES, 1999). The test item was tested in two independent experiments at several concentrations. Each assay was conducted with and without metabolic activation (S9 mix). The concentrations, including the controls were tested in triplicate. The reported data of this mutagenicity test shows that the test item did not induce gene mutations by base pair changes or frameshift in the genome of the strains used. The study has been used as key study in the NONS dossier for the source substance and has been used to conclude on the hazard assessment and classification. Therefore, the data are considered to be reliable and adequate for assessment of the hazards of the source substance. No Klimisch scores have been assigned by the original registrants and little detail is included in the robust study summary provided for the source substance but the study is GLP-compliant, guideline study, and therefore is considered reliable.

Both the studies have been used as key studies in the registration dossier for the source substances and have been used to conclude on the hazard assessment and classification of the substance. Therefore, the data are considered to be reliable and adequate source studies.

 

The in vitro cytogenicity of the substance reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide was assessed in a GLP-compliant study following OECD guideline 473 (LPT 2018a). The study was conducted using human lymphocyte cultures with and without metabolic activation (S9 mix). The main test was conducted in three independent experiments with four test concentrations. Two replicate cultures were used at each concentration tested. Under the test conditions, reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide, tested up to the maximum feasible concentration of 500 μg/mL medium that led to test item precipitation and/or cytotoxicity in the absence and in the presence of metabolic activation, revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage. Positive controls (mitomycin C and cyclophosphamide) induced significant damages, which confirmed the validity of this assay. The study is GLP-compliant, guideline study, and has been assigned a Klimisch score of 1 and is considered to be a reliable and adequate study to complete the cytogenicity endpoint.

 

The potential of the substance reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide to induce gene mutations was examined in an in vitro study according to OECD 490 guideline (LPT 2018b). The study was conducted using cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation (S9 mix). The test item, tested up to the maximum feasible concentration of 500 μg/mL that led to test item precipitation and/or cytotoxicity in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. The positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions. No change was noted in the ratio of small to large mutant colonies. Therefore, test item did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that the test item, tested up to the maximum feasible concentration of 500 μg/mL, that led to test item precipitation and/or cytotoxicity, neither induced mutations nor had any chromosomal aberration potential. The study is GLP-compliant, guideline study, and has been assigned a Klimisch score of 1 and is considered to be a reliable and adequate study for the genetic toxicity endpoint.

Justification for classification or non-classification

Negative results were produced in an in vitro reverse mutation test in bacterial cells with two read across substances, dilithium azelate and reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide.

 

No indications of mutagenic properties with respect to chromosomal or chromatid damage were observed in the in vitro cytogenicity in mammalian cells study with the substance reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide tested up to the maximum feasible concentration of 500 μg/mL, that led to test item precipitation and/or cytotoxicity in the absence and in the presence of metabolic activation. The test item has not induced mutations nor had any chromosomal aberration potential in the in vitro gene mutation assay in mammalian cells.

Therefore, no classification is considered relevant for this endpoint for substance reaction mass of amines, hydrogenated tallow alkyl and azelaic acid and lithium hydroxide.