Nonanamide, N,N-dimethyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May 2017 to 8 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Repeated Dose 28-Day Oral Toxicity Study in Rodents” adopted on 3 October 2008

Deviations:

no

Principles of method if other than guideline:

The study included recovery animals.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

N,N-dimethylnonanamide

EC Number:

612-975-5

Cas Number:

6225-08-7

Molecular formula:

C11H23NO

IUPAC Name:

N,N-dimethylnonanamide

Details on test material:

- Name of test material (as cited in study report): N,N-Dimethylnonanamide
- Synonyme: Genagen PA

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: 112-2016-11 D
- Expiration date of the batch: May 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool and dry (+2 to +8°C)
- Solubility of the test substance in the vehicle: miscible with corn oil

FORM AS APPLIED IN THE TEST: Liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the recommended species by regulatory agencies for conducting preclinical toxicological studies among rodent species

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks; The dosing was initiated not later than 8 weeks of age
- Housing: In a standard polypropylene cage (size: L 430 x B 285 x H 150 mm)
- Diet (e.g. ad libitum): Teklad Certified (2014SC) Global 14 % Protein Rodent Maintenance Diet - Pellet (Manufactured by Envigo)
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to ultraviolet rays in aquaguard water filter cum purifier

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7°C to 23.9°C
- Humidity (%): 46 to 69%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered through oral route using stainless steel gavage cannula as it is the probable route in humans

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle: Corn oil was used as vehicle for the preparation of formulations as the test item is miscible with corn oil as evidenced by the in-house solubility/ suspendibility test results
- Concentration in vehicle: G1: 0 mg/mL
G2: 100 mg/mL
G3: 300 mg/mL
G4: 1000 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose samples were taken in duplicates sets (5 mL for each set from top, middle and bottom layers) during week 1 and week 4 and were analyzed for dose concentration by the validated GC-FID system. The results were found to be within the acceptable range of 85 to 115% to the nominal concentration and less than 10.0% RSD.

Duration of treatment / exposure:

28 consecutive days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

5 males and 5 females for the dose levels of 0, 100, 300 and 1000 mg/kg bw.
5 males and 5 females for the dose levels of 0 and 1000 mg/kg bw for the recovery groups.

Control animals:

yes, concurrent vehicle

Details on study design:

Doses selected for the study were 100, 300 and 1000 mg/kg body weight as low, mid and high doses respectively. The control animals were dosed with vehicle alone. This dose levels were selected based on the results of 14-day dose-range finding study (BIO-TX 2075). There were no clinical signs or mortality were observed up to the dose of 1000 mg/kg bw. Hence, in this study, dose levels of 100, 300 and 1000 mg/kg body weight were selected- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
All animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS:
The animals were subjected to detailed clinical examinations before initiation of the treatment and at weekly intervals thereafter during the experiment. These observations were made outside the home cage and preferably at the same time on the day of examination. Signs noted included, but not limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions autonomic activity such as lacrimation, piloerection, pupil size and unusual respiratory pattern.

BODY WEIGHT:
Individual animal body weights were recorded on the Day 1, before test item administration and at weekly intervals thereafter. Fasting body weight of all the animals was recorded at terminal sacrifice.

FOOD CONSUMPTION
Individual animal food consumption was recorded weekly, coinciding with the body weights of the respective animals. Food intake per rat (g/rat/day) was calculated using the amount of food given and left over in each cage and the number of rats in each cage.

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic examination was performed for all the animals before start of the treatment and during week 4 of treatment for G1 and G4 group animals and during week 6 for recovery group animals. As the results of examinations during week 4 did not reveal any treatment related occular changes, the examination was not extended to lower main dose groups.

HAEMATOLOGY AND CLINICAL CHEMISTRY:
Blood samples were collected from all the rats of main groups on day 29 and from recovery group animals on day 43. The animals were fasted overnight before blood collection. Water was provided ad libitum during fasting period. Blood samples were collected from the animals separately into the tubes containing K2-EDTA and lithium heparin tubes for hematology and clinical chemistry respectively, Sodium citrate tubes for Prothrombin time and activated partial thromboplastin time parameters. Blood samples were collected from retro-orbital plexus puncture under mild isoflurane anaesthesia with the help of a fine capillary tube.

URINALYSIS: Yes / No / Not specified
Urine was collected from all rats of main group animals and recovery group animals on day 29 and 43 respectively. The animals were kept in urine collection cages for overnight and were fasted but water was provided ad libitum during this period. The overnight urine volume (mL) collected from these animals was measured. The urine was analyzed using “Urine analyzer” DIRUI H-500” (Dirui Industrial Company Ltd.) and analyzed for blood, bilirubin, urobilinogen, ketones, protein, glucose and leucocytes.
The volume of urine collected (mL), appearance and the color were recorded by physical evaluation.
In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
Neurological/Functional examination was carried out during week 4 for control and high dose main group animals and during last week for recovery group animals. As there were no treatment related effects, the examination was not carried out for lower dose groups.

Sacrifice and pathology:

GROSS PATHOLOGY:
At the end of treatment (main groups) and recovery period (recovery groups), all animals were fasted overnight (water allowed) and on the day of necropsy (day 29 for main groups, day 43 for recovery groups), all animals were weighed and then subjected to necropsy. The animals were euthanized by using excess CO2 anaesthesia followed by exsanguination. The gross pathological examination was carried out for each rat. Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues. The animals were sacrificed in a serial block sequence and complete gross pathological examination was performed.

HISTOPATHOLOGY:
Histopathological examination was conducted on the tissues from the vehicle control and high dose sacrificed at termination. All organs and tissue samples as defined in the study plan were processed, embedded in paraffin, cut at a thickness of 4 to 5 micrometers and stained with hematoxylin and eosin. The bone marrow smear was fixed in methanol and stained with Giemsa Stain.
The examination was not extended to the lower dose groups and recovery groups as there were no treatment related effects noted at the high dose.

Statistics:

The raw data was subjected to statistical analysis. The computer printout of the data (in the form of appendix) was verified with the original raw data. After verification, the data was subjected to statistical analyses using SPSS software version 22. Body weight, percent change in body weight, feed consumption, organ weights and ratios, hematological and clinical chemistry estimations and urine analysis parameters (pH, specific gravity and urobilinogen) were subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test was done for different treatment groups comparing with the control group data. Comparison of means between the recovery groups was done using ‘t’ test based on the requirement. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The values obtained for high dose animals towards end ot the treatment were reduced for females sacrificd at the termination of the treatment. However, oppositive tendency was found for females sacrificed after recovery period. This finding was considered to be incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

effects observed, non-treatment-related

Behaviour (functional findings):

effects observed, non-treatment-related

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

no effects observed

Description (incidence and severity):

Not applicable

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect found up to the highese dose of 1000 mg/kg bw/day

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1-1:SUMMARY OF MAEL BODY WEIGHTS (g)

Dose (mg/kg body weight/day)		Days
		1	8	15	22	28	36	42
Animals sacrificed on day 29
0	Mean	148.73	189.19	229.37	268.41	286.09
	±SD	15.02	22.45	28.51	37.14	40.33
	n	5	5	5	5	5
100	Mean	147.62	186.97	226.20	262.88	285.75
	±SD	14.06	14.01	16.90	18.48	23.39
	n	5	5	5	5	5
300	Mean	149.43	181.02	223.26	262.20	287.66
	±SD	11.71	18.86	23.00	24.23	25.73
	n	5	5	5	5	5
1000	Mean	150.63	182.30	222.18	256.17	271.66
	±SD	12.30	17.51	19.93	16.21	19.35
	n	5	5	5	5	5
Animals sacrificed on day 43 (recovery animals)
0	Mean	150.60	188.38	230.55	260.49	289.73	320.41	346.68
	±SD	5.62	11.02	17.47	18.63	21.04	20.89	19.80
	n	5	5	5	5	5	5	5
1000	Mean	148.93	180.65	220.21	248.55	276.29	305.82	323.82
	±SD	10.36	12.57	22.94	29.23	32.64	37.18	41.23
	n	5	5	5	5	5	5	5

M: Male; F: Female; SD: Standard Deviation; n: Number of animals

Table 1 -2:SUMMARY OF FEMAEL BODY WEIGHTS (g)

Dose (mg/kg body weight/day)		Days
		1	8	15	22	28	36	42
Animals sacrificed on day 29
0	Mean	138.98	165.33	191.89	215.17	222.24
	±SD	9.29	10.52	12.11	13.48	10.94
	n	5	5	5	5	5
100	Mean	135.93	157.98	180.40	197.95	208.89
	±SD	12.10	13.77	16.53	15.43	20.30
	n	5	5	5	5	5
300	Mean	136.63	151.88	173.78	184.36*	200.89
	±SD	8.28	14.36	16.70	12.24	18.93
	n	5	5	5	5	5
1000	Mean	139.20	154.15	170.29	187.34*	196.61
	±SD	6.41	7.82	8.66	14.87	18.36
	n	5	5	5	5	5
Animals sacrificed on day 43 (recovery animals)
0	Mean	139.61	162.33	187.15	206.54	217.44	235.08	247.34
	±SD	5.51	8.33	13.09	15.60	14.86	16.57	18.37
	n	5	5	5	5	5	5	5
1000	Mean	139.52	167.56	196.34	218.90	234.41	254.69	266.41
	±SD	4.11	5.83	7.30	8.09	7.75	11.97	12.86
	n	5	5	5	5	5	5	5

M: Male; F: Female; SD: Standard Deviation; n: Number of animals

Table 2-1: SUMMARY OF HAEMATOLOGY RECORDFOR MALES

Dose (mg/kg body weight/day)		Total Leucocyte Count              (WBC)              (10^3cells/µL)	Total Erythrocyte Count             (RBC)               (10^6cells/µL)	Hemoglobin (HGB)  (g/dL)	Haematocrit (HCT)                   (%)	Mean Corpuscular Volume (MCV)                  (fL)	Mean Corpuscular Hemoglobin (MCH)                      (pg)	Mean Corpuscular Haemoglobin Concentration (MCHC)                (g/dL)	Platelet Count               (PLT)                (10^3cells/µL)	Mean Platelet Volume            (MPV)                 (fL)
Animals sacrificed on day 29
0	Mean	11.24	7.24	12.98	41.82	57.82	17.96	31.04	810.60	5.68
	±SD	1.71	0.36	0.19	1.03	1.75	0.69	0.42	78.30	0.19
	n	5	5	5	5	5	5	5	5	5
100	Mean	10.12	6.97	12.74	40.26	57.74	18.30	31.70	836.20	5.72
	±SD	2.42	0.12	0.34	1.30	1.58	0.49	0.55	46.83	0.22
	n	5	5	5	5	5	5	5	5	5
300	Mean	11.06	7.01	12.74	40.06	57.20	18.20	31.82*	781.60	5.96
	±SD	3.99	0.46	0.72	2.17	1.18	0.45	0.22	91.29	0.35
	n	5	5	5	5	5	5	5	5	5
1000	Mean	11.31	7.46	13.44	42.40	56.82	17.98	31.66	885.20	6.08
	±SD	2.81	0.28	0.74	1.61	0.54	0.44	0.58	191.70	0.28
	n	5	5	5	5	5	5	5	5	5
Animals sacrificed on day 43 (recovery animals)
0	Mean	9.54	7.37	13.58	41.40	56.16	18.40	32.82	910.80	6.36
	±SD	1.15	0.28	0.58	1.66	1.13	0.67	0.66	89.66	0.17
	n	5	5	5	5	5	5	5	5	5
1000	Mean	7.52	7.53	13.70	42.34	56.24	18.20	32.38	800.60	6.38
	±SD	1.92	0.36	0.49	1.83	1.07	0.56	0.60	92.08	0.18
	n	5	5	5	5	5	5	5	5	5

SD: Standard Deviation; n: number of animals

*. The mean difference is significant at the 0.05 level.

Table 2-2: SUMMARY OF HAEMATOLOGY RECORD FOR MALES

Dose (mg/kg body weight/day)		Absolute Reticulocyte Count                  (10^9cells/L)	Absolute Neutrophils              (10^3cells/µL)	Absolute Lymphocytes              (10^3cells/µL)	Absolute Monocytes              (10^3cells/µL)	Absolute Eosinophils              (10^3cells/µL)	Absolute Basophils              (10^3cells/µL)	Prothrombin Time                      (Seconds)	Activated Prothrombin Time                       (Seconds)
Animals sacrificed on day 29
0	Mean	159.90	2.24	8.33	0.41	0.07	0.02	16.82	17.18
	±SD	25.09	0.60	1.54	0.16	0.03	0.01	0.54	1.30
	n	5	5	5	5	5	5	5	5
100	Mean	150.40	1.77	7.75	0.34	0.07	0.03	16.14	16.20
	±SD	36.29	0.29	2.03	0.14	0.02	0.01	0.81	1.47
	n	5	5	5	5	5	5	5	5
300	Mean	149.82	1.63	8.69	0.49	0.04	0.03	14.12*	17.20
	±SD	31.36	0.73	3.69	0.17	0.01	0.01	0.89	1.94
	n	5	5	5	5	5	5	5	5
1000	Mean	111.90	1.61	8.77	0.58	0.13	0.03	15.92	18.48
	±SD	23.75	0.21	2.54	0.23	0.16	0.02	1.94	2.92
	n	5	5	5	5	5	5	5	5
Animals sacrificed on day 43 (recovery animals)
0	Mean	137.54	1.67	7.42	0.24	0.06	0.01	19.60	23.70
	±SD	31.22	0.65	1.48	0.05	0.01	0.01	4.31	9.83
	n	5	5	5	5	5	5	5	5
1000	Mean	114.44	1.42	5.64	0.28	0.05	0.01	17.36	32.60
	±SD	32.77	0.39	1.50	0.12	0.02	0.01	0.80	9.24
	n	5	5	5	5	5	5	5	5

SD: Standard Deviation; n: number of animals

*. The mean difference is significant at the 0.05 level.

Table 2 -3: SUMMARY OF HAEMATOLOGY RECORDFOR FEMALES

Dose (mg/kg body weight/day)		Total Leucocyte Count              (WBC)              (10^3cells/µL)	Total Erythrocyte Count             (RBC)               (10^6cells/µL)	Hemoglobin (HGB)  (g/dL)	Haematocrit (HCT)                   (%)	Mean Corpuscular Volume (MCV)                  (fL)	Mean Corpuscular Hemoglobin (MCH)                      (pg)	Mean Corpuscular Haemoglobin Concentration (MCHC)                (g/dL)	Platelet Count               (PLT)                (10^3cells/µL)	Mean Platelet Volume            (MPV)                 (fL)
Animals sacrificed on day 29
0	Mean	7.51	6.96	12.58	38.60	55.42	18.10	32.68	895.00	5.94
	±SD	0.74	0.26	0.29	1.29	1.14	0.56	0.50	62.05	0.36
	n	5	5	5	5	5	5	5	5	5
100	Mean	9.84	7.37	13.30	41.62	56.48	18.08	31.98	887.80	6.36
	±SD	0.94	0.43	0.56	2.20	1.47	0.65	0.55	99.61	0.19
	n	5	5	5	5	5	5	5	5	5
300	Mean	9.17	7.20	12.72	40.04	55.62	17.66	31.80*	843.80	6.70*
	±SD	2.85	0.41	0.90	2.66	1.34	0.54	0.32	375.79	0.72
	n	5	5	5	5	5	5	5	5	5
1000	Mean	8.82	7.47	13.44	42.08*	56.28	17.98	31.94	937.80	6.26
	±SD	1.73	0.33	0.74	1.99	0.86	0.41	0.50	36.70	0.15
	n	5	5	5	5	5	5	5	5	5
Animals sacrificed on day 43 (recovery animals)
0	Mean	8.80	7.33	13.34	40.16	54.78	18.18	33.20	1028.20	6.48
	±SD	1.12	0.43	0.81	2.82	1.06	0.33	0.51	163.39	0.33
	n	5	5	5	5	5	5	5	5	5
1000	Mean	8.13	7.07	13.34	39.54	55.98	18.88	33.70	930.20	6.44
	±SD	0.64	0.17	0.59	1.59	1.54	0.59	0.31	223.14	0.09
	n	5	5	5	5	5	5	5	5	5

SD: Standard Deviation; n: number of animals

*. The mean difference is significant at the 0.05 level.

Table 2 -4: SUMMARY OF HAEMATOLOGY RECORDFOR FEMALES

Dose (mg/kg body weight/day)		Absolute Reticulocyte Count                  (10^9cells/L)	Absolute Neutrophils              (10^3cells/µL)	Absolute Lymphocytes              (10^3cells/µL)	Absolute Monocytes              (10^3cells/µL)	Absolute Eosinophils              (10^3cells/µL)	Absolute Basophils              (10^3cells/µL)	Prothrombin Time                      (Seconds)	Activated Prothrombin Time                       (Seconds)
Animals sacrificed on day 29
0	Mean	90.22	1.51	5.49	0.28	0.10	0.01	13.40	20.26
	±SD	25.34	0.39	0.31	0.05	0.06	0.00	0.70	3.04
	n	5	5	5	5	5	5	5	5
100	Mean	92.70	2.06	7.21	0.34	0.09	0.02	13.56	18.72
	±SD	5.06	0.83	0.60	0.11	0.04	0.00	0.25	4.25
	n	5	5	5	5	5	5	5	5
300	Mean	129.14*	1.81	6.84	0.31	0.10	0.02	14.12	17.78
	±SD	21.95	0.48	2.46	0.12	0.08	0.01	1.34	2.75
	n	5	5	5	5	5	5	5	5
1000	Mean	111.18	1.60	6.76	0.27	0.06	0.01	15.80*	16.02
	±SD	21.84	0.69	1.35	0.11	0.02	0.01	1.01	3.32
	n	5	5	5	5	5	5	5	5
Animals sacrificed on day 43 (recovery animals)
0	Mean	112.42	1.31	7.04	0.24	0.09	0.02	16.26	28.20
	±SD	39.08	0.30	0.79	0.11	0.02	0.01	1.28	10.18
	n	5	5	5	5	5	5	5	5
1000	Mean	96.46	1.59	5.98*	0.32	0.13	0.01	17.56	32.50
	±SD	15.54	0.29	0.42	0.04	0.05	0.00	1.55	12.87
	n	5	5	5	5	5	5	5	5

SD: Standard Deviation; n: number of animals

*. The mean difference is significant at the 0.05 level.

Table 3-1: SUMMARY OF CLINICAL CHEMISTRY RECORDFOR MALES

Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Calcium
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)	(ALB)	(ALT)	(AST)	(ALP)	(CAL)
		mg/dL	mg/dL	mg/dL	mg/dL	mg/dL	g/dL	g/dL	U/L	U/L	U/L	mg/dL
Animals sacrificed on day 29
0	Mean	103.20	22.36	0.51	62.60	80.60	6.80	3.29	66.20	120.60	428.20	10.38
	±SD	13.48	4.01	0.03	12.62	26.88	0.28	0.18	11.84	22.70	80.00	0.44
	n	5	5	5	5	5	5	5	5	5	5	5
100	Mean	98.20	13.96*	0.49	71.80	95.80	6.46	3.24	64.20	115.20	401.20	10.12
	±SD	6.38	4.40	0.03	14.91	30.15	0.17	0.07	22.07	28.27	56.25	0.28
	n	5	5	5	5	5	5	5	5	5	5	5
300	Mean	104.00	14.90*	0.47	66.00	77.20	6.56	3.28	58.60	109.00	429.40	9.78
	±SD	10.75	3.53	0.05	7.07	32.43	0.18	0.18	7.50	6.44	112.97	0.33
	n	5	5	5	5	5	5	5	5	5	5	5
1000	Mean	108.00	13.82*	0.47	78.60	70.60	6.76	3.40	70.20	110.80	433.00	10.18
	±SD	11.47	3.02	0.04	11.55	8.88	0.23	0.08	6.22	8.58	66.85	0.41
	n	5	5	5	5	5	5	5	5	5	5	5
Animals sacrificed on day 43 (recovery animals)
0	Mean	96.40	18.92	0.51	47.60	62.40	6.46	3.05	47.80	85.20	171.40	8.86
	±SD	10.29	4.60	0.04	11.17	23.92	0.13	0.13	3.56	6.18	31.28	0.18
	n	5	5	5	5	5	5	5	5	5	5	5
1000	Mean	96.40	20.66	0.54	49.20	55.80	6.54	3.13	51.60	90.40	178.80	8.78
	±SD	9.21	6.11	0.02	8.58	14.04	0.32	0.14	9.76	6.19	39.80	0.22
	n	5	5	5	5	5	5	5	5	5	5	5

SD: Standard Deviation; n: number of animals; *. The mean difference is significant at the 0.05 level.

Table 3-2: SUMMARY OF CLINICAL CHEMISTRY RECORD FOR MALES

Dose (mg/kg body weight/day)		Phosphorous	Globulin	Albumin/Globulin ratio	Blood Urea Nitrogen	Cholinesterase	Total Bilirubin	Sodium	Potassium	Chloride
		(PHO)	(GLO)	(A/G Ratio)	(BUN)	(CHE)	(BIT)	(Na)	(K)	(CLO)
		mg/dL	g/dL	mg/dL	mg/dL	U/L	mg/dL	mmol/L	mmol/L	mmol/L
Animals sacrificed on day 29
0	Mean	7.58	3.51	0.95	10.44	158.00	0.00	154.56	4.83	113.20
	±SD	0.66	0.35	0.12	1.87	23.89	0.00	1.06	0.30	2.16
	n	5	5	5	5	5	5	5	5	5
100	Mean	7.34	3.22	1.01	6.52*	163.60	0.00	153.06	4.74	111.42
	±SD	0.53	0.20	0.08	2.05	30.06	0.00	1.68	0.19	1.10
	n	5	5	5	5	5	5	5	5	5
300	Mean	7.54	3.28	1.01	6.95*	152.60	0.02*	150.12*	4.89	109.06*
	±SD	0.52	0.26	0.12	1.65	11.44	0.02	2.67	0.21	1.86
	n	5	5	5	5	5	5	5	5	5
1000	Mean	7.44	3.36	1.01	6.45*	153.60	0.01	154.24	4.86	112.72
	±SD	0.55	0.21	0.07	1.41	31.67	0.01	2.58	0.28	1.88
	n	5	5	5	5	5	5	5	5	5
Animals sacrificed on day 43 (recovery animals)
0	Mean	5.94	3.41	0.90	8.83	174.00	0.00	143.32	3.47	103.88
	±SD	0.13	0.11	0.06	2.15	27.18	0.01	0.98	0.09	1.33
	n	5	5	5	5	5	5	5	5	5
1000	Mean	5.78	3.41	0.93	9.64	167.00	0.02	143.12	3.58	104.90
	±SD	0.34	0.34	0.12	2.85	27.22	0.02	0.47	0.18	1.26
	n	5	5	5	5	5	5	5	5	5

SD: Standard Deviation; n: number of animals; *. The mean difference is significant at the 0.05 level.

Table 3-3: SUMMARY OF CLINICAL CHEMISTRY RECORD FOR FEMALES

Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Calcium
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)	(ALB)	(ALT)	(AST)	(ALP)	(CAL)
		mg/dL	mg/dL	mg/dL	mg/dL	mg/dL	g/dL	g/dL	U/L	U/L	U/L	mg/dL
Animals sacrificed on day 29
0	Mean	113.40	24.36	0.50	62.20	49.40	6.98	3.66	41.80	97.00	160.40	11.10
	±SD	7.40	2.65	0.03	6.98	8.96	0.31	0.14	9.50	9.19	25.34	0.36
	n	5	5	5	5	5	5	5	5	5	5	5
100	Mean	124.00	19.32	0.48	66.20	64.60	6.68	3.71	43.40	96.00	183.80	10.66
	±SD	21.04	8.48	0.04	10.47	17.29	0.13	0.05	9.99	14.14	44.20	0.65
	n	5	5	5	5	5	5	5	5	5	5	5
300	Mean	112.00	18.30	0.45	74.80	60.20	6.74	3.54	49.80	112.00	188.20	10.24
	±SD	15.72	5.21	0.05	6.06	12.97	0.38	0.12	11.05	28.37	12.05	0.61
	n	5	5	5	5	5	5	5	5	5	5	5
1000	Mean	102.40	18.40	0.46	75.80*	52.20	7.28	3.80	52.60	96.60	261.40	10.04*
	±SD	6.84	4.10	0.05	7.26	16.41	0.23	0.12	4.56	17.78	148.07	0.66
	n	5	5	5	5	5	5	5	5	5	5	5
Animals sacrificed on day 43 (recovery animals)
0	Mean	103.80	20.96	0.57	52.00	47.80	6.66	3.23	42.00	92.60	129.20	8.98
	±SD	6.69	5.23	0.03	1.87	13.63	0.35	0.09	5.96	9.21	54.05	0.29
	n	5	5	5	5	5	5	5	5	5	5	5
1000	Mean	101.40	24.04	0.56	61.60*	72.20	6.38	3.30	36.00	90.80	105.80	9.06
	±SD	5.18	4.28	0.06	7.37	24.48	0.16	0.18	5.52	7.33	28.70	0.50
	n	5	5	5	5	5	5	5	5	5	5	5

SD: Standard Deviation; n: number of animals; *. The mean difference is significant at the 0.05 level.

Table 3-4: SUMMARY OF CLINICAL CHEMISTRY RECORD FOR FEMALES

Dose (mg/kg body weight/day)		Phosphorous	Globulin	Albumin/Globulin ratio	Blood Urea Nitrogen	Cholinesterase	Total Bilirubin	Sodium	Potassium	Chloride
		(PHO)	(GLO)	(A/G Ratio)	(BUN)	(CHE)	(BIT)	(Na)	(K)	(CLO)
		mg/dL	g/dL	mg/dL	mg/dL	U/L	mg/dL	mmol/L	mmol/L	mmol/L
Animals sacrificed on day 29
0	Mean	6.96	3.32	1.11	11.37	513.00	0.03	152.40	4.79	112.38
	±SD	0.30	0.23	0.07	1.24	200.57	0.03	2.75	0.41	2.78
	n	5	5	5	5	5	5	5	5	5
100	Mean	7.00	2.97	1.25*	9.02	380.00	0.01	153.86	4.83	114.60
	±SD	0.48	0.15	0.07	3.96	98.16	0.01	3.18	0.35	3.75
	n	5	5	5	5	5	5	5	5	5
300	Mean	7.08	3.20	1.12	8.54	309.20*	0.01	153.34	5.01	112.34
	±SD	0.29	0.33	0.11	2.43	29.52	0.02	1.88	0.43	2.28
	n	5	5	5	5	5	5	5	5	5
1000	Mean	7.32	3.48	1.10	8.59	476.00	0.02	155.30	4.76	115.44
	±SD	0.94	0.19	0.07	1.91	85.69	0.02	1.56	0.33	1.29
	n	5	5	5	5	5	5	5	5	5
Animals sacrificed on day 43 (recovery animals)
0	Mean	4.08	3.43	0.95	9.78	418.60	0.00	142.30	3.56	103.96
	±SD	2.01	0.28	0.06	2.44	105.36	0.03	1.39	0.28	1.12
	n	5	5	5	5	5	5	5	5	5
1000	Mean	5.38	3.08	1.08	11.22	454.80	0.02	143.30	3.61	104.38
	±SD	0.53	0.27	0.15	2.00	148.08	0.04	1.61	0.30	2.69
	n	5	5	5	5	5	5	5	5	5

SD: Standard Deviation; n: number of animals; *. The mean difference is significant at the 0.05 level.

Table 4 -1: SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g) FOR MALES

Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Epididymes/ Uterus	Testes/ Ovaries	Heart	Kidneys	Brain	Liver	Prostate+Seminal vesicles with coagulating glands (PSC)
Animals sacrificed on day 29
0	Mean	0.0483	0.4606	0.5708	0.9083	3.0201	1.0835	2.0651	1.9668	10.5234	1.8954
	±SD	0.0034	0.0943	0.1500	0.1120	0.1713	0.1911	0.3345	0.1332	1.9888	0.3913
	n	5	5	5	5	5	5	5	5	5	5
100	Mean	0.0488	0.4182	0.5165	0.8639	2.9240	1.1796	2.1493	1.9938	10.7583	1.8386
	±SD	0.0071	0.0757	0.1086	0.1179	0.5206	0.2400	0.1715	0.0709	0.7062	0.4282
	n	5	5	5	5	5	5	5	5	5	5
300	Mean	0.0520	0.4351	0.6032	0.8777	3.0423	1.0541	2.2062	2.0253	11.5609	1.9436
	±SD	0.0109	0.0856	0.0976	0.0489	0.2532	0.1404	0.2607	0.0387	1.6337	0.4910
	n	5	5	5	5	5	5	5	5	5	5
1000	Mean	0.0543	0.4018	0.5382	0.9725	3.1035	0.9934	2.0214	2.0664	11.5706	1.8636
	±SD	0.0090	0.0890	0.0517	0.1163	0.3229	0.1047	0.2636	0.0997	1.3157	0.2603
	n	5	5	5	5	5	5	5	5	5	5
Animals sacrificed on day 43 (recovery animals)
0	Mean	0.0515	0.5546	0.6454	1.2785	3.1981	1.2739	2.8651	2.0476	11.3674	2.7965
	±SD	0.0047	0.0700	0.0436	0.0742	0.0610	0.1003	0.3773	0.1157	0.6824	0.2325
	n	5	5	5	5	5	5	5	5	5	5
1000	Mean	0.0561	0.4934	0.5703*	1.2227	3.1419	1.2951	2.5595	2.0857	10.3506*	3.1057
	±SD	0.0103	0.0392	0.0256	0.1144	0.2042	0.1153	0.4456	0.0620	0.6893	0.4037
	n	5	5	5	5	5	5	5	5	5	5

SD: Standard Deviation; n: number of animals; *. The mean difference is significant at the 0.05 level.

Table 4-2: SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g) FOR FEMALES

Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Uterus	Ovaries	Heart	Kidneys	Brain	Liver
Animals sacrificed on day 29
0	Mean	0.0604	0.4292	0.4767	0.4349	0.1345	0.9491	1.5955	1.9396	7.9994
	±SD	0.0114	0.0936	0.0541	0.0869	0.0447	0.1087	0.1312	0.0399	0.6383
	n	5	5	5	5	5	5	5	5	5
100	Mean	0.0627	0.5004	0.4412	0.4209	0.1373	0.8667	1.5871	1.9428	8.0021
	±SD	0.0074	0.0651	0.0457	0.0950	0.0214	0.0616	0.1050	0.0308	0.2780
	n	5	5	5	5	5	5	5	5	5
300	Mean	0.0554	0.4624	0.4608	0.4575	0.1240	0.8119	1.6024	1.8825	7.9831
	±SD	0.0069	0.0980	0.0806	0.1626	0.0183	0.1242	0.2708	0.0834	1.0254
	n	5	5	5	5	5	5	5	5	5
1000	Mean	0.0588	0.3895	0.4226	0.3980	0.1203	0.7775*	1.3864	1.8554	8.1730
	±SD	0.0095	0.0278	0.0338	0.0605	0.0225	0.0700	0.1037	0.0892	0.6379
	n	5	5	5	5	5	5	5	5	5
Animals sacrificed on day 43 (recovery animals)
0	Mean	0.0646	0.4612	0.5441	0.6062	0.1627	0.9888	1.9524	2.0064	8.0022
	±SD	0.0082	0.0468	0.1081	0.0789	0.0291	0.0827	0.2423	0.0759	0.3885
	n	5	5	5	5	5	5	5	5	5
1000	Mean	0.0802*	0.5168*	0.5357	0.5486	0.2238	1.0916	1.8898	1.9589	8.9898
	±SD	0.0120	0.0229	0.0874	0.1217	0.0713	0.1160	0.1645	0.0543	1.3322
	n	5	5	5	5	5	5	5	5	5

SD: Standard Deviation; n: number of animals; *. The mean difference is significant at the 0.05 level.

Applicant's summary and conclusion

Conclusions:

The repeated dose toxicity of the registration substance Genagen PA/N.N-Dimethylnonamide was evaluated according to the Guideline OECD 407. No significant effect was found up to the highest dose of 1000 mg/kg bw. The NOAEL of 1000 mg/kg bw/day was obtained.

Executive summary:

The repeated dose toxicity of the registration substance Genagen PA/N.N-Dimethylnonamide was evaluated according to the Guideline OECD 407.

Rats were treated for 28 days per gavage, the applied doses being 0, 100, 300 and 1000 mg/kg bw/day. The study included recovery animals that were treated at doses of 0 or 1000 mg/kg bw/day for 28 days and were allowed to recover for 14 days.

The observation parameters included clinical signs, body weight, food consumption, functional observation batteries, ophthalmoscopic examination, hematology, clinical chemistry, urine analysis, macroscopic pathology and histophathology.

No significant effect was found up to the highest dose of 1000 mg/kg bw. The NOAEL of 1000 mg/kg bw/day was obtained.