Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Discussion of mutagenicity on the substance itself and analogues.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 August 2013 - 19 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).Strains Genotype Type of mutations indicatedTA1537 his C 3076; rfa-; uvrB-: frame shift mutationsTA98 his D 3052; rfa-; uvrB-;R-factor : frame shift mutationsTA1535 his G 46; rfa-; uvrB-: base-pair substitutionsTA100 his G 46; rfa-; uvrB-;R-factor : base-pair substitutionsEscherichia coliStrain Genotype Type of mutations indicatedWP2uvrA trp-; uvrA-: base-pair substitution
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/B-Naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
The vehicle control used was tetrahydrofuran.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
Test Item
The test item was immiscible in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but was fully miscible in tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. Tetrahydrofuran was therefore selected as the vehicle.The test item was accurately weighed and approximate half-log dilutions prepared in tetrahydrofuran by mixing on a vortex mixer on the day of each experiment. Tetrahydrofuran is toxic to the bacterial cells at and above 50 µL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 µL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981). All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicatepellets with a nominal pore diameter of 4 x 10-4 microns.Control ItemsVehicle and positive controls were used in parallel with the test item. The vehicle control used was tetrahydrofuran.
The positive control items used in the series of plates without S9-mix were as follows
:N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 µg/plate for WP2uvrA
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 3 µg/plate for TA100
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 5 µg/plate for TA1535
9-Aminoacridine (9AA): 80 µg/plate for TA1537
4-Nitroquinoline-1-oxide (4NQO): 0.2 uµg/plate for TA9

8In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the absence of metabolizing enzymes, were used in the series of plates with S9-mix at the following concentrations:
2-Aminoanthracene (2AA): 1 µg/plate for TA100
2-Aminoanthracene (2AA): 2 µg/plate for TA1535 and TA1537
2-Aminoanthracene (2AA): 10 µg/plate for WP2uvrA
Benzo(a)pyrene (BP): 5 µg/plate for TA98

Microsomal Enzyme Fraction
Lot No. PB/BNF S9 14 July 2013 was used in this study. The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/B-Naphthoflavone at80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed bycentrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 deg C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.The study was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedures) Act 1986. The conduct of the study may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process.S9-Mix and AgarThe S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL1.65 M KCl/0.4 M MgCl2 1.0 mL0.1 M Glucose-6-phosphate 2.5 mL0.1 M NADP 2.0 mL0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL Sterile distilled water14.5 mLA 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.Top agar was prepared using 0.6% Bacto agar (lot number 2299373 09/17) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 34989 09/13 and 35101 10/13).

Test Procedure
Test for Mutagenicity (Experiment 1 - Range-Finding Test) – Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.025 mL of the appropriate concentration of test item or vehicle or 0.1 mL of appropriate positive control was added to 2 mL of trace amino-acid supplemented media (at approximately 45 deg C) containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Each concentration of the test item, appropriate positive control, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the trace amino-acid supplemented media instead of phosphate buffer.All of the plates were incubated at 37 deg C± 3 deg C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity (Experiment 2 - Main Test) – Pre-Incubation Method
As Experiment 1 (the range-finding test) was deemed negative, Experiment 2 (main test) was performed using the pre-incubation method in the presence andabsence of metabolic activation.
Dose selection
The dose range used for Experiment 2 (main test) was determined by the results of Experiment 1 (the range-finding test) and was 50 to 5000 µg/plate.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.025 mL of the test item formulation or solvent or 0.1 mL of appropriate positive control were incubated at37 deg C± 3 deg C for 20 minutes (with shaking) prior to addition of 2 mL of amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 deg C± 3 deg C for 20 minutes (with shaking) and addition of amino-acid supplemented media. All testing for this experiment was performed in triplicate.All of the plates were incubated at 37 deg C± 3 deg C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the General Study Plan, Section 2.2.2 (negative controls). Combined historical negative and solvent control ranges for 2011 and 2012 are presented in Appendix 2.All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive control reference items for 2011 and 2012 are presented within the report.There should be a minimum of four non-toxic test item dose levels. There should be no evidence of excessive contamination.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).2. A reproducible increase at one or more concentrations.3. Biological relevance against in-house historical control ranges.4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
As above
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 (range-finding test) and Table 4 and Table 5for Experiment 2 (main test) of the report. The results are also expressed graphically in Figure 1 to Figure 4 of the report. Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare are presented in Appendix 2 of the report. A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 2.The maximum dose level of the test item in the range-finding test was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test and consequently the same maximum dose level was used in the second mutation test. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test. A test item precipitate (globular in appearance) was observed at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the range-finding test (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the main test (pre-incubation method). All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Tabulated data and figures are appended below.

Conclusions:
Interpretation of results (migrated information):negative NovaSpec 450 was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the range-finding test was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test and consequently the same maximum dose level was used in the second mutation test. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test. A test item precipitate (globular in appearance) was observed at 5000 ug/plate, this observation did not prevent the scoring of revertant colonies. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the range-finding test (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the main test (pre-incubation method).

NovaSpec 450 was considered to be non-mutagenic under the conditions of this test.

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

Genetic toxicity in vivo

Description of key information

Discussion of mutagenicity on the substance itself

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 October 2014 to 18 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Crl:WI rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony. Males and females originated from different units to avoid subsequent brother/sister matings.
Hygienic level: Standard laboratory conditions during the study
Justification of species/strain: The rat is regarded as a suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for dose range finding study (study code:14/328-220PE).
Number of animals: 48 male, 48 female rats, 12 animals/sex/group, 4 groups (main animals); 10 male and 10 female rats, 5/sex/group (recovery group) and 12 males and 12 females rats in the positive control group for the micronucleus test).
Age of animals: Young adult rats, 10-11 weeks old at starting and 12-13 weeks at mating.
Body weight range: Males: 327 g – 478 g, Females: 207 g - 273 g;
Acclimation period: At least 5 days
Husbandry
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.
Room number: 508
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® and GRADE 5 type wooden chips were available to the animals
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1 – 25.1 °C (target range 22±3°C)
Relative humidity: 31 – 64 % (target range 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting)
.Food and water supply: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.

Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36, Hungary). The quality control results are retained in the archives at CiToxLAB Hungary Ltd.Food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Animal identification: Each parental animal (P Generation) was identified by a number unique within the study written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd. The animal number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section. The boxes (cages) were marked by identity cards, with information at least about study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery. Boxes were arranged in such a way that possible effects due to cage placement were minimized.Randomization: All adult animals were sorted according to body weight by computer and divided in to weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that the mean group weights were similar. The grouping was controlled by SPSS/PC software, according to the actual body weight verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.
Route of administration:
oral: gavage
Vehicle:
Name: Poly(ethylene glycol) 400
Lot No.: BCBL5307V/BCBM8497V//BCBK 9981V
Manufacturer: Sigma-Aldrich Co.
Expiry Date: 31 January 2015/31 January 2016/January 2015
Storage: Room temperature
Name: 0.2% Polysorbate 80Lot No.: BCBL9041V/BCBN3690V
Manufacturer: Sigma-Aldrich Co.
Expiry Date: 30 November 2014/31 July 2016
Storage: Room temperature under inert gas
Preparation of the vehicle: For the preparation of 100 g (approximately 100 mL) vehicle, 0.2 g Polysorbate 80 was weighed by analytical balance into 99.8 g PEG 400, and was stirred by a magnetic stirrer.
Details on exposure:
The test item was formulated in the vehicle as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared for 7 days and kept in refrigerator pending dosage in the first 14 days of the study, and daily afterwards.The positive control material (cyclophosphamide) was dissolved in physiological saline (10 mg/mL) for the treatment. The solution was prepared just before the treatment. The test item solutions were given to assure the same dosing volumes in rat (2 mL/kg bw).No dose formulation analysis was performed from the positive control solutions.
Duration of treatment / exposure:
At least until the first scheduled euthanasia of dams.
Frequency of treatment:
Daily
Post exposure period:
At least 14 days after the first schedules euthanasia of dams
Remarks:
Doses / Concentrations:0, 1000 mg/kg bw/dayBasis:nominal conc.
No. of animals per sex per dose:
5 male and 5 female in the treatment group12 animals/sex/group served as the positive control group for the Mammalian Erythrocyte Micronucleus Test (MNT).
Control animals:
yes
Positive control(s):
12 animals/sex/group served as the positive control group for the Mammalian Erythrocyte Micronucleus Test (MNT). They were mated and femalesallowed to deliver similarly to the Main animals, then treated once with 20 mg/kg bw Cyclophosphamide, administered by intraperitoneal injection(2 mL/kg bw) approximately 24 h prior to scheduled necropsy.
Tissues and cell types examined:
Polychromatic erythrocytes (PCEs) were scored per animal
Details of tissue and slide preparation:
At the end of the treatment period, bone marrow slides were prepared from all animals in the vehicle control and the positive control groups. The bone marrow was obtained from the right femurs of the rats immediately after euthanasia and flushed with foetal bovine serum (5 mL). The left femur of Main and Recovery group animals was used for routine histopathology, the left femur of positive control animals was discarded.After vortex mixing, the cell suspension was concentrated by centrifugation and the supernatant was discarded. Smears of the cell pellet were made on four standard microscope slides. Slides were then dried at room temperature until considered to be completely dry. Subsequently the slides were stained as follows:1. Fixed for a minimum of 5 minutes in methanol and allowed to air-dry.2. Stained with 10% Giemsa solution for 20 minutes.3. Rinsed in distilled water.4. Dried at room temperature (at least 12 hours).5. Coated with EZ-mountingPrior to sending to Microptic for microscopic analysis, one slide from each animal was given a code number for blind microscopic analysis. The code labels covered the original animal numbers to ensure that the slides were scored without bias.
Evaluation criteria:
Two thousand Polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of the micronucleated cells based on the first 2000 PCEs counted in the optic field. The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by also counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs). During this process, the number of micronuclei was recorded in mature erythrocytes (NCEs) as well. Criteria for Identification of Micronucleated ErythrocytesA micronucleus is defined in following way:- A bluish mauve strongly coloured uniform round or oval particle in the cell.- The particle should be large enough for the colour to be recognisable, and it should be located inside the cells. Areas with micronucleus-like particles outside the cells should not be used for analysis.- During focusing, the particle should stay uniform in colour/light refraction and shape within a large interval and focus in the same plane as the erythrocyte.- The unit of damage is deemed to be the cell, and therefore cells with two or more micronuclei will be counted as single micronucleated cells. The Micronucelus Test is considered acceptable/valid in the conditions of this study, as it met the following criteria:- the frequencies of micronucleated polychromatic erythrocytes found in the negative and/or solvent controls fell within the range of historical laboratory control data.- the positive control item produced biologically relevant increases in the number of micronucleated polychromatic erythrocytes. - Each treated and control group included at least 5 analysable animals.
Statistics:
Statistical analysis was performed using Kruskal Wallis Non Parametric ANOVA test (level of significance 5%)
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The groups with 1000 mg/kg bw (high dose) were compared with their vehicle control group using Kruskal Wallis test. These gave values of H = 0.014 in the males and H = 0.149 in the females. Both H values are non-significant, giving a negative response. The positive and negative control results were also compared, and gave a value of H = 17.633 (p<0.001) in the males, and H = 17.561 (p<0.001) in the females. The positive control treatment therefore caused a very substantial increase, demonstrating the sensitivity of the test system. The positive and negative control data are considered to give adequate data to confirm the validity of the study.

Table 1: Dose Group – Males – Negative Control

Animal no.

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 NCE+PCE

1001

84

2

310

1002

101

3

291

1003

99

3

360

1004

43

3

356

1005

104

1

382

1006

26

3

305

1007

107

1

460

1008

5

2

372

1009

79

1

217

1010

16

1

432

1011

28

3

308

1012

22

1

380

Mean

 

2.00

347.8

SD

 

0.95

66.0

 

Table 2: Dose Group – Males – High Dose 1000 mg/kg bw 24h

Animal no.

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 NCE+PCE

4001

106

2

361

4002

98

2

395

4003

116

5

370

4004

103

4

295

4005

25

2

336

4006

83

1

451

4007

56

2

425

4008

13

1

384

4009

54

4

391

4010

90

0

208

4011

36

1

369

4012

50

1

380

Mean

 

2.08

363.8

SD

 

1.51

62.9

 

Animal no.

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 NCE+PCE

5001

24

7

213

5002

29

9

316

5003

33

11

481

5004

55

7

305

5005

111

7

200

5006

118

12

297

5007

113

10

215

5008

114

6

388

5009

15

10

221

5010

120

13

239

5011

44

32

395

5012

11

8

269

Mean

 

11.00

294.9

SD

 

6.97

88.0

 

Table 4: Dose Group – Females – Negative Control

Animal no.

Slide code

Micronucleated PCE/2000 PCE

PCE/1000

NCE+PCE

1501

20

3

601

1502

37

2

484

1503

70

1

536

1504

14

2

537

1505

32

1

544

1506

45

2

397

1507

10

0

511

1508

35

2

420

1509

1

3

409

1510

30

2

507

1511

19

5

509

1512

80

2

479

Mean

 

2.08

494.5

SD

 

1.24

60.8

 

Table 5: Dose Group – Females – High Dose 1000 mg/kg bw 48h

Animal no.

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 NCE+PCE

4501

77

4

552

4502

62

1

589

4503

81

2

417

4504

4

1

554

4505

65

0

364

4506

109

2

489

4507

94

1

500

4508

74

0

556

4509

21

6

477

4510

88

3

419

4511

72

1

399

4512

57

5

486

Mean

 

2.17

483.5

SD

 

19.5

71.6

 

Table 6: Dose Group – Females - Cyclophosphamide

Animal no.

Slide code

Micronucleated PCE/2000 PCE

PCE/1000 NCE+PCE

5501

47

30

464

5502

75

52

569

5503

2

42

544

5504

87

35

424

5505

59

36

516

5506

49

101

592

5507

9

27

497

5508

38

162

522

5509

41

34

416

5510

96

11

271

5511

52

61

429

5512

34

37

399

Mean

 

52.33

470.3

SD

 

41.08

89.0

 

Conclusions:
Interpretation of results: negativeIn conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of NovaSpec Base Oil to rats at up to and including 1000 mg/kg bw. Thus there was no evidence of any genotoxic activity of the test item under the conditions of this part of the study.
Executive summary:

The objective of this work phase is to assess the potential genotoxic effects of the test item by examining the induction of micronuclei in bone marrow erythrocytes of treated and control animals.

 

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of NovaSpec Base Oil to rats at up to and including 1000 mg/kg bw. Thus there was no evidence of any genotoxic activity of the test item under the conditions of this part of the study.

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

Additional information

Additional information from genetic toxicity in vitro:  

Genetic toxicity is assessed using a combination of available study data and information on analogous oils. The following are available for inspection:

 

NovaSpec 450: Reverse Mutation Assay 'Ames Test' using Salmonella typhimurium and Escherichia coli

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the range-finding test (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the main test (pre-incubation method).

 

NovaSpec 450 was considered to be non-mutagenic under the conditions of this test.

Effects of hydrocarbons on transformation and intercellular communication in Syrian hamster embryo cells.

The main purpose of the study was to test the potential of different hydrocarbons to induce or promote morphological transformation and inhibit intercellular communication in primary Syrian hamster embryo cells. None of the hydrocarbons utilised in the assessment induced morphological transformation in Syrian hamster embryo cells. Co-exposure of the hydrocarbons and benzo(a)pyrene resulted in the enhancement of transformation frequency in certain compounds only. Evaluation of the results indicates that for the iso-alkanes, the enhancement decreases with increasing number of carbon atoms. This decrease may be due to reduced water solubility, and thus a reduction in the solubilized concentration.

 

In the case of the substance to be registered, the iso-alkane carbon range lies between C15 and C60 (approximately). On the basis of this study, it is deemed that the likelihood of morphological transformation of cells is minimal, and hence associated mutagenicity due to cell transformation is unlikely. No effects are therefore proposed.

 

NovaSpec Base Oil: Genetic toxicity in vitro-Chromosome Aberration Test

NovaSpec base oil did not induce a significant level of chromosome aberrations in the performed experiments with or without metabolic activation using Chinese hamster V79 lung cells.. Therefore, NovaSpec base oil is considered not clastogenic in this test system

 

NovaSpec Base Oil: Genetic toxicity in vitro-Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay)

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of NovaSpec base oil to cause gene mutation and/or chromosome damage. The results of which concluded that no biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. Therefore is deemed to not be mutagenic under the conditions of the study.

 

NovaSpec Base Oil: Genetic toxicity in vivo-Mammalian Erythrocyte Micronucleus Test (MNT)

No induction of micronuclei in bone marrow erythrocytes was observed following administration of the test substance to rats at up to and including 1000 mg/kg bw. Thus there was no evidence of any genotoxic activity of the test item under the conditions of this study.

 

Genetic toxicity of high-boiling petroleum substances

The purpose of inclusion of this literature paper in support of the registration of this substance is to provide peer reviewed evidence of the lack of mutagenicity of oil based products that are similar to the substance. As discussed above, the paper is designed to draw an analogy between likely mutagenicity and the presence of PAC’s. However, as can be seen in section 1, the synthesis of the substance subject to the registration does not result in the formation of PAC’s as these are absent from the synthetic route, and hence result in a “cleaner” product.

 

The literature paper contains a detailed summary of the mutagenicity evaluations from a number of studies. Cytogenetic tests for chromosome aberrations and micronucleus formation have produced generally negative results for HBPS. Many samples (e.g. catalytically cracked clarified oil) selected for testing were considered most likely to be biologically active based on systemic toxicity in repeated dose studies, mutagenicity in bacteria and mammalian cells and content of aromatic hydrocarbons, yet did not induce cytogenetic damage in bone marrow. The ability of these substances to reach target organs was verified by effects on haematology parameters and bone marrow. Although a few samples have produced positive results the most reasonable general conclusion is that most HBPS do not produce chromosomal aberrations when tested under in vivo conditions in current assays. Negative results from in vitro studies in CHO cells suggest that negative results in vivo are not consequences of lack of exposure of the organ being evaluated (i.e. bone marrow) but are true indicators of the absence of clastogenic activity for these substances. There was no relationship between results in the Optimized Ames Salmonella test and cytogenetic assays. This observation is consistent with the general genetic toxicology literature, most recently in Clarke et al. (2012) and U.S. EPA which specifies the need to include both gene mutation and cytogenetic testing to establish a complete genetic toxicity profile.

 

As such, it is considered appropriate to utilise this paper in support of the fact that the base oils of this type are proposed to not induce mutagenicity in mammalian cells. The substance subject to registration, by weight of evidence, is also not proposed to induce such effects. 

 

Justification for selection of genetic toxicity endpoint 

GLP Study

Justification for classification or non-classification

A Klimish one rated GLP study conducted in accordance with current OECD guidelines plus associated literature data is available to assess genetic toxicity. The overall interpretation is that the results are negative. White mineral oils do not have a history of mutagenic potential. This effect is not applicable to this category of substances.

No classification is applicable.