Butanoic acid, 3-oxo-, 1,1'-[2-[(1,3-dioxobutoxy)methyl]-2-ethyl-1,3-propanediyl] ester

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06 January 2023 – 28 January 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 407

Deviations:

yes

Remarks:

(The study period was only 14 days as it was conducted as a dose range finding study for a subsequent more extensive toxicity study)

GLP compliance:

no

Remarks:

DRF study performed without GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

422-630-9

EC Name:

-

Cas Number:

22208-25-9

Molecular formula:

C18H26O9

IUPAC Name:

2,2-bis({[(3-oxobutanoyl)oxy]methyl})butyl 3-oxobutanoate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks (at receipt)
- Weight at study initiation: 213.78-217.06 g (range of average values of each group of males); 179.99-183.89 g (range of average values of each group of females)
- Fasting period before study: No
- Housing: Maximum of three animals of same sex were housed in a standard polycarbonate cage (Size: L 43 X B 28 X H 21 cm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized corn cob was provided as bedding material.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the acclimatization and experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
Analyses of feed and water were performed before starting the study. The corresponding certificates are annexed to the test report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1-22.9 ºC
- Humidity (%): 46-66 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 06 January 2023 To: 28 January 2023

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared before dose administration and homogeneity was achieved by thorough stirring using a magnetic stirrer during administration. The required quantity of test item was weighed separately and transferred, mixed and stirred using glass rod and thereafter the entire quantity of the formulation was transferred into measuring cylinder. The rinsing procedure was repeated (many times). Finally, the volume was adjusted to required mark in measuring cylinder with vehicle to get a desired concentration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): In a solubility test, the test item formed suspension in corn oil. Hence, corn oil was selected as vehicle to prepare test item formulations in this study. Corn oil is one of the commonly used vehicles in toxicology studies.

- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): not reported.
- Purity: N/A

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item in dose formulations was not established under this dose range finding study. However, freshly prepared test item formulations were administered to the animals and homogeneity was achieved by thorough stirring using magnetic stirrer. The actual dose volume for each animal was calculated based on the most recent body weight.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once a day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 - Vehicle control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2 - Low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3 - Mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4 - High dose

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses of 0, 100, 300 and 1000 mg/kg bw/day have been selected for dose range finding study as no literature is available for the test item.
- Fasting period before blood sampling for clinical biochemistry: The animals were fasted overnight before blood collection. Water was provided ad libitum during fasting period.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weight was recorded on the day of randomization, on day of treatment (Day 1) prior to treatment and weekly thereafter.

FOOD CONSUMPTION: yes
-feed consumption was measured at weekly intervals. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of necropsy (day 15)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all animals
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by coagulation analyzer.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy (day 15)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all animals
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Triglycerides, Phosphorous, Calcium, Blood Urea Nitrogen, Globulin, Albumin/Globulin ratio. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Prolyte Na/K/Cl analyzer (Medica Corporation).

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of necropsy (day 15)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Leucocytes. 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.

Sperm parameters (parental animals):

Testes and epididymides were subjected to necropsy and detailed gross pathological examination. These organs were weighed and weight ratios as percentage of body weight were determined.

Postmortem examinations (parental animals):

SACRIFICE
-On day15, all animals of Group G1, G2, G3 and G4 were subjected to necropsy and detailed gross pathological examination

GROSS NECROPSY
-Gross pathological examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, as well as organs and tissues of each animal with special emphasis on reproductive organs.
The following organs from all animals at the scheduled sacrifices were weighed wet as soon as possible to avoid drying: Kidneys, Adrenals, Spleen, Heart, Liver, Thymus, Brain, Lungs Testes/Ovaries, Epididymides/Uterus, Prostate along with seminal vesicles with coagulating gland.

HISTOPATHOLOGY / ORGAN WEIGHTS
-The above mentioned organs from all animals were collected, weighed and preserved. The organ weight ratios as percentage of body weight were determined.

Statistics:

After verification, the data was subjected to statistical analysis using SPSS software, version 27. Body weight, percent change in body weight with respect to day 1, haematological, clinical chemistry estimations parameters absolute and relative organ weights 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. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were seen in all the tested dose group animals in either sex.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any dose group during the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related variations in mean body weight and percent body weight change were noted, when compared to vehicle control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was not any impact of test item on feed consumption in any of the tested group in either sex.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse test item related changes in the haematology and coagulation parameters. However, in males statistically significant (p<0.05) increases in Erythrocyte count (RBC), Haemoglobin concentration (HGB), Haematocrit (HCT) Eosinophils in G4 was noted and in females decrease in Prothrombin Time (PT) in G2 and G4 was noted. The observed variation is considered incidental in the absence of dose responsiveness and similar changes were not noted in other sex.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related adverse changes were observed in any of the clinical chemistry parameters, in both sexes when compared to the vehicle control.
However, in G2 female statistically significant (p<0.05) increase in glucose was noted. The noted variation lacked dose responsiveness, hence is considered incidental.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No gross pathological changes or effects in absolute and relative weights were noted in testes or epididymides at any dose tested.

Reproductive performance:

not examined

Details on results (P0)

No gross pathological changes or effects in absolute and relative weights were noted in reproductive organs at any dose tested.

Effect levels (P0)

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 treatment related effects found up to highest tested dose of 1000 mg/kg bw/d.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

Effect levels (F1)

Remarks on result:

other: F1 was not assessed as this study was conducted as a dose range finding study for a subsequent more extensive toxicity study

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1. Summary of clinical signs of toxicity, detailed clinical examination and mortality record

Group, Sex & Dose (mg/kg body weight/day)	No. of Animals	Clinical Signs of Toxicity (No of animals exhibiting signs/Total no. of animals)	Mortality (No. of Incidences/ No. of Animals)
G1, M & 0	3	N	0/3
G2, M & 100	3	N	0/3
G3, M & 300	3	N	0/3
G4, M & 1000	3	N	0/3
G1, F & 0	3	N	0/3
G2, F & 100	3	N	0/3
G3, F & 300	3	N	0/3
G4, F & 1000	3	N	0/3

 

Table 2. Summary of body weight (g) record

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Days
		1	8	14
G1, M & 0	Mean	215.52	236.13	258.22
	±SD	27.54	33.11	34.05
	n	3	3	3
G2, M & 100	Mean	215.55	241.01	264.64
	±SD	13.93	17.21	20.88
	n	3	3	3
G3, M & 300	Mean	213.78	237.88	257.62
	±SD	11.77	13.98	25.67
	n	3	3	3
G4, M & 1000	Mean	217.06	240.54	259.24
	±SD	5.56	6.47	10.16
	n	3	3	3
G1, F & 0	Mean	180.83	193.18	207.07
	±SD	11.46	9.00	6.98
	n	3	3	3
G2, F & 100	Mean	183.89	193.86	210.85
	±SD	13.30	15.13	15.73
	n	3	3	3
G3, F & 300	Mean	179.99	193.78	202.25
	±SD	5.34	3.54	3.17
	n	3	3	3
G4, F & 1000	Mean	183.35	197.98	211.12
	±SD	5.14	2.66	9.26
	n	3	3	3

 

Table 3. Summary of percent change in body weight (%) with respect to day 1 record

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Days
		1 to 8	1 to 14
G1, M & 0	Mean	9.44	19.77
	±SD	2.03	0.50
	n	3	3
G2, M & 100	Mean	11.81	22.75
	±SD	2.66	4.23
	n	3	3
G3, M & 300	Mean	11.30	20.53
	±SD	3.72	10.27
	n	3	3
G4, M & 1000	Mean	10.86	19.52
	±SD	4.18	6.62
	n	3	3
G1, F & 0	Mean	6.91	14.66
	±SD	2.02	3.72
	n	3	3
G2, F & 100	Mean	5.39	14.75
	±SD	0.60	6.28
	n	3	3
G3, F & 300	Mean	7.75	12.47
	±SD	4.86	5.07
	n	3	3
G4, F & 1000	Mean	8.02	15.18
	±SD	2.45	5.08
	n	3	3

 

Table 4. Summary of feed consumption (g/rat/day) record

Group, Sex & Dose (mg/kg body weight/day)		Week 1	Week 2
G1, M & 0	Mean	19.01	19.59
	±SD	-	-
	n	1	1
G2, M & 100	Mean	19.64	22.03
	±SD	-	-
	n	1	1
G3, M & 300	Mean	20.34	20.40
	±SD	-	-
	n	1	1
G4, M & 1000	Mean	21.02	20.49
	±SD	-	-
	n	1	1
G1, F & 0	Mean	16.99	16.83
	±SD	-	-
	n	1	1
G2, F & 100	Mean	15.12	15.99
	±SD	-	-
	n	1	1
G3, F & 300	Mean	16.82	17.44
	±SD	-	-
	n	1	1
G4, F & 1000	Mean	16.64	17.46
	±SD	-	-
	n	1	1

 

Table 5. Summary of gross pathology findings

Sex	Male				Female
Group	G1	G2	G3	G4	G1	G2	G3	G4
Dose (mg/kg body weight/day)	0	100	300	1000	0	100	300	1000
Number of animals	3	3	3	3	3	3	3	3
No. of dead rats during treatment	0	0	0	0	0	0	0	0
No. of moribund sacrificed rats	0	0	0	0	0	0	0	0
No. of terminally sacrificed rats	3	3	3	3	3	3	3	3
No of rats showing gross pathology	0	0	0	0	0	0	0	0

 

Table 6. Summary of haematology record

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Hemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Hemoglobin	Mean Corpuscular Hemoglobin Concentration
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)
G1, M & 0	Mean	15.41	7.66	15.03	44.63	58.30	19.63	33.63
	±SD	3.40	0.18	0.58	2.12	2.84	0.95	0.49
	n	3	3	3	3	3	3	3
G2, M & 100	Mean	20.59	7.85	15.50	47.43	60.53	19.83	32.73
	±SD	3.47	0.31	0.46	0.50	2.85	1.24	0.60
	n	3	3	3	3	3	3	3
G3, M & 300	Mean	11.31	7.88	15.53	47.27	59.93	19.70	32.87
	±SD	3.65	0.11	0.45	2.15	2.28	0.36	0.64
	n	3	3	3	3	3	3	3
G4, M & 1000	Mean	13.96	8.61*	16.37*	49.90*	57.97	19.00	32.77
	±SD	2.38	0.14	0.51	1.25	0.61	0.46	0.57
	n	3	3	3	3	3	3	3
G1, F & 0	Mean	14.09	7.75	14.87	43.47	56.10	19.20	34.23
	±SD	2.26	0.80	1.25	4.44	0.35	0.44	0.85
	n	3	3	3	3	3	3	3
G2, F & 100	Mean	14.20	7.76	15.57	43.93	56.73	20.20	35.63
	±SD	5.77	0.93	0.58	4.95	3.25	1.81	2.89
	n	3	3	3	3	3	3	3
G3, F & 300	Mean	13.26	7.26	14.77	41.33	56.90	20.43	35.93
	±SD	5.42	0.75	0.59	4.48	1.11	1.36	2.55
	n	3	3	3	3	3	3	3
G4, F & 1000	Mean	12.50	7.65	14.93	44.07	57.63	19.47	33.83
	±SD	1.75	0.41	0.57	2.06	1.17	0.31	0.55
	n	3	3	3	3	3	3	3

 

Group, Sex & Dose (mg/kg body weight/day)		Platelet Count	Mean Platelet Volume	Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(PLT)	(MPV)	(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(10^3 cells/µL)	(fL)	(%)	(%)	(%)	(%)	(%)	(%)
G1, M & 0	Mean	713.00	7.47	4.04	15.50	78.80	3.10	1.00	0.40
	±SD	464.46	0.38	1.21	5.30	6.46	1.30	0.50	0.10
	n	3	3	3	3	3	3	3	3
G2, M & 100	Mean	946.00	7.53	4.37	18.50	75.77	2.43	1.17	0.47
	±SD	239.95	0.21	0.71	14.04	14.26	0.75	1.24	0.15
	n	3	3	3	3	3	3	3	3
G3, M & 300	Mean	913.33	7.73	2.96	18.10	71.87	5.80	2.23	0.30
	±SD	88.64	0.21	0.61	0.60	0.38	1.31	1.02	0.00
	n	3	3	3	3	3	3	3	3
G4, M & 1000	Mean	994.67	7.53	3.23	24.10	65.77	4.53	4.00*	0.33
	±SD	104.27	0.21	0.86	2.46	2.21	1.89	1.01	0.06
	n	3	3	3	3	3	3	3	3
G1, F & 0	Mean	937.00	7.60	3.42	19.10	74.00	3.77	1.20	0.33
	±SD	307.15	0.20	2.00	3.89	2.89	1.21	0.46	0.06
	n	3	3	3	3	3	3	3	3
G2, F & 100	Mean	764.67	8.20	3.40	16.30	75.63	2.87	3.20	0.43
	±SD	361.51	0.50	1.52	7.47	10.83	1.65	2.31	0.06
	n	3	3	3	3	3	3	3	3
G3, F & 300	Mean	896.00	7.93	4.09	19.03	72.40	3.87	2.13	0.40
	±SD	162.86	0.25	1.84	5.69	5.96	0.50	1.33	0.17
	n	3	3	3	3	3	3	3	3
G4, F & 1000	Mean	985.67	8.13	3.85	22.40	71.70	3.13	1.53	0.27
	±SD	65.52	0.15	0.28	4.26	4.79	0.75	0.59	0.06
	n	3	3	3	3	3	3	3	3

 

Group, Sex & Dose (mg/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute  Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^9 cells/L)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(Seconds)	(Seconds)
G1, M & 0	Mean	308.97	2.30	12.24	0.48	0.14	0.06	17.33	17.37
	±SD	90.40	0.51	3.50	0.21	0.06	0.04	1.25	0.84
	n	3	3	3	3	3	3	3	3
G2, M & 100	Mean	343.43	3.53	15.89	0.50	0.21	0.10	16.36	20.59
	±SD	62.23	2.11	5.20	0.15	0.21	0.04	0.57	1.98
	n	3	3	3	3	3	3	3	3
G3, M & 300	Mean	233.13	2.06	8.12	0.62	0.28	0.03	16.27	18.77
	±SD	49.68	0.73	2.60	0.04	0.22	0.02	0.77	2.34
	n	3	3	3	3	3	3	3	3
G4, M & 1000	Mean	277.90	3.33	9.21	0.63	0.57	0.05	16.61	16.25
	±SD	75.02	0.32	1.76	0.30	0.23	0.01	1.36	1.57
	n	3	3	3	3	3	3	3	3
G1, F & 0	Mean	254.47	2.71	10.41	0.51	0.17	0.05	17.62	19.53
	±SD	122.89	0.86	1.54	0.13	0.05	0.02	0.61	4.33
	n	3	3	3	3	3	3	3	3
G2, F & 100	Mean	261.13	2.07	11.05	0.39	0.39	0.06	15.72*	17.88
	±SD	118.33	0.40	5.73	0.19	0.18	0.02	0.42	5.81
	n	3	3	3	3	3	3	3	3
G3, F & 300	Mean	297.23	2.39	9.70	0.53	0.28	0.06	16.50	18.70
	±SD	145.43	0.64	4.50	0.29	0.17	0.05	0.82	3.98
	n	3	3	3	3	3	3	3	3
G4, F & 1000	Mean	294.43	2.75	9.02	0.38	0.18	0.03	16.09*	21.79
	±SD	18.86	0.12	1.84	0.09	0.05	0.02	0.36	0.81
	n	3	3	3	3	3	3	3	3

*: Statistically significant (P<0.05)

 

Table 7. Summary of clinical chemistry record

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)	(ALB)	(ALT)	(AST)	(ALP)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	g/dL)	(g/dL)	(U/L)	(U/L)	(U/L)
G1, M & 0	Mean	101.67	29.37	0.55	58.33	66.67	7.20	3.22	53.33	121.67	221.33
	±SD	10.60	5.35	0.02	4.93	17.04	0.20	0.10	4.93	24.19	74.10
	n	3	3	3	3	3	3	3	3	3	3
G2, M & 100	Mean	96.33	29.53	0.57	62.00	55.33	7.50	3.30	68.33	129.67	236.33
	±SD	7.64	5.85	0.02	12.77	13.87	0.30	0.17	3.21	7.23	54.50
	n	3	3	3	3	3	3	3	3	3	3
G3, M & 300	Mean	89.67	29.80	0.52	51.67	101.33	7.03	3.33	61.67	122.33	341.67
	±SD	4.16	5.20	0.01	6.43	65.96	0.21	0.16	9.07	20.55	89.40
	n	3	3	3	3	3	3	3	3	3	3
G4, M & 1000	Mean	97.00	29.00	0.54	55.67	59.33	7.33	3.45	58.33	113.33	328.33
	±SD	4.36	5.55	0.04	8.50	35.73	0.49	0.17	9.07	6.03	32.65
	n	3	3	3	3	3	3	3	3	3	3
G1, F & 0	Mean	86.33	39.23	0.57	62.00	72.67	7.67	3.38	55.00	127.00	247.00
	±SD	4.16	6.38	0.04	7.81	33.65	0.06	0.09	9.00	17.78	90.54
	n	3	3	3	3	3	3	3	3	3	3
G2, F & 100	Mean	104.67*	35.70	0.60	65.67	61.67	7.70	3.46	99.33	181.33	182.33
	±SD	4.51	10.36	0.03	16.44	32.13	0.35	0.24	68.07	58.14	60.34
	n	3	3	3	3	3	3	3	3	3	3
G3, F & 300	Mean	95.33	29.93	0.54	54.67	75.33	7.37	3.26	57.00	139.33	232.00
	±SD	8.08	4.66	0.03	5.13	39.88	0.25	0.20	4.58	17.39	164.79
	n	3	3	3	3	3	3	3	3	3	3
G4, F & 1000	Mean	98.67	24.57	0.53	73.00	66.67	7.33	3.52	55.00	106.33	204.33
	±SD	5.86	0.90	0.01	22.65	23.86	0.50	0.22	2.65	11.02	55.37
	n	3	3	3	3	3	3	3	3	3	3

 

Group, Sex & Dose (mg/kg body weight/day)		Total Bilirubin	Calcium	Phosphorous	Cholinesterase	Globulin	Albumin/Globulin ratio	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(BIT)	(CAL)	(PHO)	(CHE)	(GLO)	(A/G Ratio)	(BUN)	(Na)	(K)	(CLO)
		(mg/dL)	(mg/dL)	(mg/dL)	(U/L)	(g/dL)	-	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, M & 0	Mean	0.11	10.03	6.13	154.67	3.98	0.81	13.73	139.60	3.71	105.57
	±SD	0.02	0.12	0.60	16.17	0.27	0.08	2.50	1.71	0.37	2.20
	n	3	3	3	3	3	3	3	3	3	3
G2, M & 100	Mean	0.07	10.10	6.05	121.00	4.20	0.79	13.80	141.87	3.93	107.47
	±SD	0.01	0.46	0.34	37.64	0.14	0.03	2.73	0.15	0.56	1.00
	n	3	3	3	3	3	3	3	3	3	3
G3, M & 300	Mean	0.11	10.13	6.12	101.00	3.71	0.90	13.92	140.10	3.65	104.30
	±SD	0.04	0.49	0.64	42.53	0.09	0.04	2.43	0.46	0.18	4.06
	n	3	3	3	3	3	3	3	3	3	3
G4, M & 1000	Mean	0.10	10.27	5.98	115.33	3.88	0.89	13.55	139.23	3.66	102.63
	±SD	0.02	0.25	0.12	11.59	0.34	0.05	2.59	2.25	0.10	3.91
	n	3	3	3	3	3	3	3	3	3	3
G1, F & 0	Mean	0.09	10.20	5.69	448.67	4.29	0.79	18.34	139.53	3.81	106.87
	±SD	0.04	0.10	0.34	218.60	0.12	0.04	2.98	1.70	0.14	1.78
	n	3	3	3	3	3	3	3	3	3	3
G2, F & 100	Mean	0.06	10.03	4.96	488.33	4.24	0.82	16.68	141.23	3.73	108.33
	±SD	0.01	0.31	1.52	401.85	0.46	0.14	4.84	1.27	0.43	1.08
	n	3	3	3	3	3	3	3	3	3	3
G3, F & 300	Mean	0.08	9.87	5.18	415.33	4.11	0.79	13.99	139.63	3.74	107.10
	±SD	0.02	0.15	0.31	199.14	0.07	0.04	2.18	1.25	0.07	1.75
	n	3	3	3	3	3	3	3	3	3	3
G4, F & 1000	Mean	0.08	9.97	5.28	362.67	3.81	0.93	11.48	140.93	3.72	105.77
	±SD	0.01	0.35	0.47	208.41	0.29	0.02	0.43	0.84	0.12	2.20
	n	3	3	3	3	3	3	3	3	3	3

*: Statistically significant (P<0.05)

 

Table 8. Summary of urinalysis record

Examination	Group, Sex & Dose (mg/kg/day)		G1, M & 0	G2, M & 100	G3, M & 300	G4, M & 1000
	Number of Animals		3	3	3	3
Physical Examination	Color	Pale Yellow	3	-	3	3
		Yellow	-	3	-	-
	Apperance	Clear	3	1	3	3
		Turbidity	-	2	-	-
	Volume (mL)	Mean	2.2	2.7	1.2	3.0
		±SD	0.8	0.3	0.3	0.5
Chemical Examination	pH	Mean	5.8	6.3	5.7	5.8
		±SD	0.3	0.3	0.3	0.3
	Specific gravity (SG)	Mean	1.030	1.030	1.030	1.030
		±SD	0.000	0.000	0.000	0.000
	Urobilinogen (UBG)               (mg/dL)	Mean	0.7	0.2	1.0	0.7
		±SD	0.5	0.0	0.0	0.5
	Bilirubin (BIL)   (mg/dL)	1	3	3	3	3
	Ketones (KET) (mg/dL)	5	3	3	3	2
		15	-	-	-	1
	Blood (BLD) (Ery/µL)	Neg	2	2	-	-
		Ca80	-	-	-	1
		Ca10	-	1	1	-
		Ca25	-	-	2	2
		>=Ca200	1	-	-	-
	Protein (PRO) (mg/dL)	30	3	3	3	3
	Nitrite (NIT)	Neg	2	-	1	2
		Pos	1	3	2	1
	Leucocytes (LEU) (Leu/µL)	Ca70	2	3	-	-
		Ca15	1	-	3	3
	Glucose (GLU) (mg/dL)	Neg	3	3	3	3
Microscopic Examination	Epi Cells	0	2	1	3	2
		0-1	1	1	-	1
		1-2	-	1	-	-
	Casts	Absent	3	3	3	3
	Crystals	Present	3	3	3	3

 

Examination	Group, Sex & Dose (mg/kg/day)		G1, F & 0	G2, F & 100	G3, F & 300	G4, F & 1000
	Number of Animals		3	3	3	3
Physical Examination	Color	Pale Yellow	3	3	2	3
		Yellow	-	-	1	-
	Apperance	Clear	3	3	2	3
		Turbidity	-	-	1	-
	Volume (mL)	Mean	1.2	2.7	2.2	4.3
		±SD	0.3	0.8	0.8	1.8
Chemical Examination	pH	Mean	6.0	5.8	7.3	5.8
		±SD	0.0	0.3	1.6	0.3
	Specific gravity (SG)	Mean	1.030	1.030	1.017	1.025
		±SD	0.000	0.000	0.012	0.005
	Urobilinogen (UBG)               (mg/dL)	Mean	0.7	0.2	0.7	0.2
		±SD	0.5	0.0	0.5	0.0
	Bilirubin (BIL)   (mg/dL)	Neg	-	1	-	2
		1	3	2	3	1
	Ketones (KET) (mg/dL)	5	3	2	3	1
		Neg	-	1	-	2
	Blood (BLD) (Ery/µL)	Neg	3	1	3	3
		Ca10	-	2	-	-
	Protein (PRO) (mg/dL)	Neg	-	-	-	3
		Trace	-	3	1	-
		>=300	-	-	1	-
		100	-	-	1	-
		30	3	-	-	-
	Nitrite (NIT)	Neg	-	2	1	3
		Pos	3	1	2	-
	Leucocytes (LEU) (Leu/µL)	Neg	-	1	1	3
		Ca70	-	-	2	-
		Ca15	3	2	-	-
	Glucose (GLU) (mg/dL)	Neg	3	3	3	3
Microscopic Examination	Epi Cells	0	2	3	2	2
		0-1	1	-	1	1
	Casts	Absent	3	3	3	3
	Crystals	Present	3	3	3	3

 

Table 9. Summary of absolute organ weights (g) record

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys	Brain	Liver	Prostate+Seminal vesicles with coagulating glands (PSC)
G1, M & 0	Mean	0.0569	0.3161	1.1578	2.9419	0.8424	0.8946	1.7430	1.6811	7.8730	1.4337
	±SD	0.0141	0.0269	0.2774	0.0317	0.1045	0.1037	0.2386	0.0548	0.9084	0.4801
	n	3	3	3	3	3	3	3	3	3	3
G2, M & 100	Mean	0.0434	0.3843	1.3946	2.8097	0.7433	0.9793	1.7929	1.8006	8.6424	1.4464
	±SD	0.0063	0.0561	0.4100	0.1846	0.0476	0.0701	0.0379	0.0975	1.0011	0.1312
	n	3	3	3	3	3	3	3	3	3	3
G3, M & 300	Mean	0.0471	0.2860	1.4546	3.2638	0.8225	0.9156	1.7204	1.8688	8.2005	1.4351
	±SD	0.0137	0.0853	0.6647	0.1690	0.0939	0.1069	0.1870	0.1451	1.4470	0.1714
	n	3	3	3	3	3	3	3	3	3	3
G4, M & 1000	Mean	0.0502	0.3586	1.0628	3.2475	0.8604	0.9275	1.7705	1.8239	7.9500	1.5484
	±SD	0.0118	0.0728	0.2475	0.3785	0.1349	0.0234	0.0264	0.0664	0.5808	0.1829
	n	3	3	3	3	3	3	3	3	3	3

 

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Ovaries with Oviduct	Uterus with cervix	Heart	Kidneys	Brain	Liver
G1, F & 0	Mean	0.0653	0.3518	1.3109	0.1014	0.5245	0.7561	1.4506	1.6489	6.9631
	±SD	0.0155	0.0988	0.5494	0.0221	0.3749	0.0214	0.1304	0.0324	0.6644
	n	3	3	3	3	3	3	3	3	3
G2, F & 100	Mean	0.0633	0.2851	1.0969	0.1151	0.4561	0.8306	1.4629	1.7280	6.8227
	±SD	0.0083	0.0685	0.1513	0.0116	0.0940	0.1027	0.0496	0.0323	0.4436
	n	3	3	3	3	3	3	3	3	3
G3, F & 300	Mean	0.0643	0.3018	1.0642	0.1184	0.4597	0.8005	1.4591	1.7184	6.7091
	±SD	0.0019	0.0199	0.3251	0.0298	0.1173	0.0900	0.0930	0.0377	0.4766
	n	3	3	3	3	3	3	3	3	3
G4, F & 1000	Mean	0.0762	0.3893	0.9916	0.1098	0.4997	0.8543	1.4356	1.7151	7.4837
	±SD	0.0182	0.1208	0.1645	0.0163	0.2168	0.0489	0.1095	0.0883	0.1878
	n	3	3	3	3	3	3	3	3	3

 

Table 10. Summary of organ weight relative to fasting body weight (%) record

Group, Sex & Dose (mg/kg body weight/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys	Brain	Liver	Prostate+Seminal vesicles with coagulating glands (PSC)
G1, M & 0	Mean	236.92	0.0241	0.1343	0.4901	1.2518	0.3581	0.3783	0.7343	0.7169	3.3246	0.5969
	±SD	26.27	0.0058	0.0161	0.1172	0.1354	0.0555	0.0327	0.0185	0.0990	0.1533	0.1560
	n	3	3	3	3	3	3	3	3	3	3	3
G2, M & 100	Mean	237.31	0.0183	0.1614	0.5973	1.1845	0.3137	0.4127	0.7574	0.7599	3.6472	0.6141
	±SD	16.88	0.0019	0.0130	0.2070	0.0292	0.0193	0.0073	0.0408	0.0385	0.4241	0.0962
	n	3	3	3	3	3	3	3	3	3	3	3
G3, M & 300	Mean	235.95	0.0199	0.1200	0.6028	1.3875	0.3533	0.3876	0.7303	0.7931	3.4581	0.6079
	±SD	22.99	0.0056	0.0249	0.2143	0.0692	0.0726	0.0121	0.0612	0.0171	0.2692	0.0328
	n	3	3	3	3	3	3	3	3	3	3	3
G4, M & 1000	Mean	238.03	0.0210	0.1501	0.4493	1.3677	0.3617	0.3902	0.7446	0.7676	3.3412	0.6493
	±SD	8.58	0.0045	0.0249	0.1199	0.1890	0.0577	0.0225	0.0342	0.0545	0.2420	0.0561
	n	3	3	3	3	3	3	3	3	3	3	3

Group, Sex & Dose (mg/kg/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries with Oviduct	Uterus with Cervix	Heart	Kidneys	Brain	Liver
G1, F & 0	Mean	181.39	0.0361	0.1930	0.7193	0.0557	0.2860	0.4168	0.7996	0.9098	3.8379
	±SD	5.09	0.0088	0.0496	0.2853	0.0110	0.1975	0.0013	0.0665	0.0434	0.3318
	n	3	3	3	3	3	3	3	3	3	3
G2, F & 100	Mean	192.11	0.0330	0.1498	0.5757	0.0598	0.2360	0.4313	0.7644	0.9024	3.5547
	±SD	11.58	0.0044	0.0423	0.1166	0.0025	0.0358	0.0288	0.0734	0.0730	0.2006
	n	3	3	3	3	3	3	3	3	3	3
G3, F & 300	Mean	182.68	0.0352	0.1651	0.5841	0.0647	0.2526	0.4388	0.7994	0.9410	3.6751
	±SD	3.97	0.0009	0.0076	0.1854	0.0152	0.0701	0.0553	0.0625	0.0302	0.2961
	n	3	3	3	3	3	3	3	3	3	3
G4, F & 1000	Mean	192.63	0.0393	0.1997	0.5158	0.0568	0.2547	0.4455	0.7456	0.8953	3.9044
	±SD	15.88	0.0070	0.0447	0.0882	0.0039	0.0876	0.0458	0.0109	0.0995	0.3622
	n	3	3	3	3	3	3	3	3	3	3

 

 

 

Applicant's summary and conclusion

Conclusions:

The NOAEL was determined to be at 1000 mg/kg bw/day in rats after 14 days of oral exposure.

Executive summary:

A dose range finding study was performed for the test substance in accordance with OECD guideline 407 in order to evaluate its toxic potential after repeated exposure and select the appropriate doses for a subsequent toxicity study. The test item was diluted in corn oil (vehicle) and added to Sprague-Dawley rats (3 per sex per dose) at doses of 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day for a period of 14 days. The vehicle and test item formulations were administered orally (gavage) at a dose volume of 10 mL/kg body weight. Freshly prepared test item formulations were administered to the animals as soon as possible after preparation.

All animals for each group were observed for clinical signs of toxicity once daily, mortality and morbidity twice daily, and body weight and feed consumption weekly. Clinical pathology (haematology, clinical chemistry analysis and urinalysis) was conducted for all the animals sacrificed at termination.  All animals were subjected to gross pathological examination and the organs were weighed, collected and preserved.

No clinical signs of toxicity or mortality were observed. No treatment related changes in mean body weight and percent change in body weight (%) with respect to day 1 and feed consumption were observed. No treatment related changes were observed in haematology, coagulation, clinical chemistry and urinalysis parameters. No treatment related changes in fasting body weights, organ weights and its ratios were noted. No gross pathological changes were observed. Specifically, no gross pathological changes or effects in absolute and relative weights were noted in reproductive organs at any dose tested.

Based on these results, the NOAEL was determined to be at 1000 mg/kg bw/day.