Partially hydrogenated β-3,7,11-trimethyldodeca-1,3,6,10-tetraene, reaction products with linear C8-C16 alpha olefin, hydrogenated.

Genetic toxicity: in vivo

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Partially hydrogenated β-3,7,11-trimethyldodeca-1,3,6,10-tetraene, reaction products with linear C8-C16 alpha olefin, hydrogenated.

EC Number:

939-894-0

Molecular formula:

Variable - substance is a UVCB

IUPAC Name:

Partially hydrogenated β-3,7,11-trimethyldodeca-1,3,6,10-tetraene, reaction products with linear C8-C16 alpha olefin, hydrogenated.

Test material form:

other: Water white liquid

Details on test material:

Name: NovaSpec base oilBatch/Lot No.: TS13732CAS number: 1472010-43-7Chemical name: Alkenes, C-10-16a-, mixed with (6E)-7,11-dimethyl-3-methylene-116,10-dodecatriene, dimers, tetramers and trimers, hydrogenatedPurity: 100%Manufacture date: 06 June 2014Expiry date: 06 June 2016Description: Water white liquidStorage condition: Controlled room temperature (15-25 °C below 70 RH%)Safety Precautions: Routine safety precautions for unknown materials (lab coat, gloves, safety glasses and face mask) were applied to assure personnel health and safety.No correction for purity of the test item was applied.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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.

Administration / exposure

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

Doses / concentrations

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.

Examinations

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%)

Results and discussion

Test results

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.