[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 May- 23 Sept 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid

EC Number:

940-877-5

Cas Number:

2241455-89-8

Molecular formula:

C9H12N2O4

IUPAC Name:

Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy and supplied by Envigo Holland
- Females nulliparous and non-pregnant: not reported
- Age at study initiation: 27 - 29 days
- Weight at study initiation: 68.9 - 103.3 g (males), 68.8 - 105.1 g (females)
- Housing: up to 5 rats of one sex per cage, in clear polysulfone solid bottomed cages, nesting material was provided inside suitable bedding bags and changed at least twice a week
- Diet: laboratory rodent diet (4 RF 21, Mucedola S.r.l., Milanese (MI), Italy), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analysis of water, diet and bedding material are kept on file at ERBC.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 May 2021 To: 23 September 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The formulation was prepared weekly according to stability data obtained from ERBC Study No. A4260, stirred for at least 15 minutes before dosing and maintained under magnetic stirring during administration.

VEHICLE
- Justification for use and choice of vehicle: corn oil, no justification provided
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in ERBC Study no. A4260 in the range from 20 to 200 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85-115%; precision CV < 10%).
Preparations of the test item were prepared as suspensions in corn oil. Concentration and homogeneity of the low and high dose level were assessed by taking six analytical aliquots in different positions. For the intermediate levels, only concentration was assessed by taking two different analytical aliquots. Each analytical aliquot was analysed separately.Concentration was evaluated as the mean of the single determinations and homogeneity as the coefficient of variation of the sextuplicate set.
In ERBC Study no. A4260, a 28 hour and 8 day stability at room temperature were verifed in the range from 20 to 200 mg/mL. According to ERBC SOPs, suspensions are considered to be stable if concentration and homogeneity, after the defned period of storage, are still acceptable (85%-115% for concentration and CV < 10% for homogeneity).
The proposed preparation procedure for the test item was checked in the range from 20 to 200 mg/mL by chemical analysis (concentration and homogeneity) in ERBC Study no. A4260 to confrm that the method was suitable. Final results for all levels were within the acceptability limits stated in ERBC SOPs for concentration (85-115%) and homogeneity (CV < 10%).
Samples of the preparations prepared on Day 1 and Week 13 were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).

Duration of treatment / exposure:

13 weeks + 4 weeks recovery period (for 5 males and 5 females of the control and high dose group, respectively)

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the Sponsor, based on the results obtained in a previous oral toxicity study in rats (OECD 422) and preliminary non-GLP study (ERBC Study No. E0590), in which no adverse effects were noted at all dose levels (100, 300 and 1000 mg/kg bw/day).
- Rationale for animal assignment: The rats were allocated to the 4 groups by computerised stratifed randomisation to give approximately equal initial group mean body weights.
- Fasting period before blood sampling for clinical biochemistry: At the end of recovery period, erroneously the animals were not fasted overnight, but they were under conditions of food deprivation in the morning, before blood collection.
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study. Observations were performed at the same time interval each day (2 - 2.5 hours post-dose).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once per week during the study from the start of treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: On the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION:
- The weight of food consumed by each cage of rats was recorded at weekly intervals starting from treatment. The group mean daily intake per rat was calculated.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the commencement of treatment and during Week 13 of treatment by means of an ophthalmoscope, and by a slit-lamp microscope, after the instillation of 0.5% Tropicamide.
- Dose groups that were examined: All (prior to treatment), control and high dose group (Week 13).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy and at the end of the recovery period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No (but under conditions of food deprivation)
- How many animals: All
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy and at the end of the recovery period.
- Animals fasted: No (but under conditions of food deprivation)
- How many animals: All
- Parameters checked in table [No.1] were examined.

PLASMA/SERUM HORMONES: Yes (Thyroid hormone determination (T3, T4 and TSH))
- Time of blood sample collection: Prior to necropsy
- How many animals: Main phase animals
- Parameters checked: Thyroid hormones T3, T4 and TSH

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during Week 12 of treatment and once during Week 4 of recovery an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength was performed. Motor activity was assessed once during Week 12 of treatment and once during Week 4 of recovery by an automated activity recording.
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER:
OESTRUS CYCLE: Yes
Time schedule: At the end of the study, just prior to necropsy, vaginal smears were taken from all surviving female animals, and the oestrous cycle phase recorded.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)

HISTOPATHOLOGY: Yes (see table 2)

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the signifcance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verifed by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modifed t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological fnding was carried out by means of a non-parametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the treatment period, rales were observed in 3 out of 10 males dosed at 1000 mg/kg bw/day. Ocular discharge or decreased activity were also seen in 2 of these males. Ocular discharge was also observed in a single male animal dosed at 100 mg/kg bw/day, while a palpable mass was described in a second male of the same group. No signifcant clinical signs were observed during recovery period. No treatment-related clinical signs were observed in the females during treatment and recovery periods.

No changes of toxicological signifcance were found at the weekly clinical examination, which included an evaluation of neurotoxicity.
Slight decreases in the number of rearing, statistically signifcant, were occasionally observed during the treatment-period in males and/or females dosed at 100, 300 and 1000 mg/kg bw/day, when compared to controls. Due to the occasional occurrence of these change and in the absence of other signs, they were not considered to be of toxicological relevance (non-treatment-related, non-adverse). (Please refer to table 5 in the "any other information on results incl. tables" section)

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two cases of unscheduled death occured.
One control male was sacrificed for humane reasons on Day 35, after swallowing the catheter. Piloerection and salivation were observed in this animal the day before humane sacrifice. At post mortem examination, this animal had oedematous consistency of salivary glands and unilateral minimal pelvic dilation in the kidney; microscopically there was a mild diffuse oedema in the surrounding connective tissue of salivary glands. Moderate proteinaceus plug was noted in the urinary bladder with uncertain relationship to the dilated renal pelvis. The cause of illness could be attributed to a misdosing

A high dose female was sacrifced for humane reasons on Day 48. Decreased activity, dyspnoea, hunched posture, rales, staining around perianal region and swollen abdomen were observed prior to sacrifce. At post mortem examination, this animal had distended gastrointestinal tract with abnormal gaseous content (stomach, duodenum, ileum, jejunum, ileum, caecum and colon), reduced size of the thymus and abnormal yellow staining of the skin in the urogenital region. Microscopically there was marked atrophy of the thymus, acinar cell hypertrophy in the pancreas, minimal, focal, mucosal ulceration of the non glandular region of the forestomach associated with inflammatory reaction in the submucosa. (The factors contributory to illness status of this female could not be determined.)

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to control animals, no treatment-related changes were noted in mean body weights and body weight gain in both genders, during the treatment and recovery periods of the study. At the end of the treatment period, there were no differences in terminal body weight in treated males and females, when compared to their controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed in food consumption in male and female animals, during the treatment and recovery periods.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No findings were detected in both eyes of all surviving animals, from control and all treated groups, when they were re-examined during Week 13 of treatment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

White blood cells were increased in males dosed at 1000 mg/kg bw/day (58%). Changes involved mainly lymphocytes and monocytes. At the end of the recovery period treated males showed leucocytes values comparable to controls, confirming complete reversibility. The other statistically signifcant differences between control and treated males (haemoglobin, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin concentration) were within the range of expected biological variation and/or not dose-related, therefore they were considered to be incidental.
There were no changes observed in coagulation paramaters. (Please refer to table 3 in the "any other information on results incl. tables" section)

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed. The statistically signifcant differences recorded between control and treated animals (alkaline phosphatase, aspartate aminotransferase, creatinine, calcium, sodium and phosphorus in males, glucose and phosphorus in females) were within the range of expected biological variation and/or not dose-related, therefore they were considered to be unrelated to treatment.
Recovery phase: No treatment-related changes were observed. In males, calcium was still slightly higher than controls. As for the dosing phase, this change was considered to be incidental. The other statistically signifcant differences recorded (alkaline phosphatase and sodium in females) were not recorded at the end of the Dosing phase, therefore they were considered to be unrelated to treatment. (Please refer to table 4 in the "any other information on results incl. tables" section)

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: .Thyroid hormones (T3, T4 and TSH). For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No relevant differences that could be considered treatment-related were observed at functional tests (sensory reactivity, landing footsplay, grip strength) performed at the end of treatment and recovery periods.
A slight statistically signifcant increase of mean landing footsplay was recorded in the males treated at 1000 mg/kg bw/day (19%) at the end of treatment period. No changes were observed at measurements performed at the end of recovery. These increases were not associated with other fndings, therefore they were not considered to be adverse. All the other statistically signifcant changes were considered to be incidental.
Motor activity measurements performed at the end of the treatment and recovery periods did not show any signifcant differences between treated animals and controls. (Please refer to table 7 in the "any other information on results incl. tables" section)

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Increases in relative (% to body weight) testes and adrenal gland weights were mainly present in high dose males, when compared to control group animals. The increase in testes weights was dose-related, reaching statistical signifcance in males receiving test item at 1000 mg/kg bw/day (+12%). Adrenal gland weight changes were not statistically signifcant. Since no macroscopic or microscopic correlation were noted for the adrenal gland and testes weight changes, these
organ weight variations were considered to be unrelated to treatment. Any other organ weight variations were within the range of expected variations in SD rats of the same age and considered incidental and unrelated to treatment.
(Please refer to table 6 in the "any other information on results incl. tables" section)

Recovery sacrifice: At the end of the recovery period, there were no treatment-related changes in the terminal body weight or in the absolute and relative organ weights. In particular, there were no organ weight changes in the testes and adrenal gland. Any organ weight variations were within the range of expected variations in SD rats of the same age and considered incidental and unrelated to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the treatment period, there were no treatment-related macroscopic fndings in males and females receiving up to and including 1000 mg/kg bw/day.
Any macroscopic observations, including the subcutaneous masses observed in a 100 mg/kg bw/day male and in a control female, were within the range of expected spontaneous fndings in SD rats of the same age considered incidental and unrelated to the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related microscopic observationsin males and females receiving up to and at 1000 mg/kg bw/day.
There were no treatment-related microscopic observations in the testes examined with PAS special stain.
There were no treatment-related effects on physiology of the oestrous cycle (oestrous, metestrus, diestrus and proestrus) in control and high dose females.
Any microscopic observations were within the range of expected spontaneous fndings in SD rats of the same age considered incidental and unrelated to the test item.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Oestrus cyle: No treatment-related anomalies were observed in the oestrous cycle of the treated females at the end of the treatment period, when compared to controls.

Thyroid hormone determination (T3, T4 and TSH): no changes were observed.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects of toxicological relevance observed.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 3: Selected haematology data (males)

Treatment Group (mg/kg bw/day)	Main study				Recovery phase
	Control	100	300	1000	Control	1000
Number	10	10	9	10	4	5
HGB (g/dL)	14.43± 0.533	13.86± *D 0.372	14.09± 0.558	14.77± 0.362	14.00± 0.245	14.08± 0.432
HCT (%)	44.59± 1.730	42.75± *D 0.925	43.97± 1.572	44.80± 0.961	42.75± 0.592	42.62± 1.632
LYM (x10^3/µL)	4.139± 1.1188	4.068± 1.2046	4.732± 0.9578	6.868± +D 1.1948	3.168± 1.0994	4.472± 1.6311
MON (x10^3/µL)	0.181± 0.0905	0.199± 0.0677	0.168± 0.0823	0.304± +D 0.0829	0.178± 0.0846	0.224± 0.0783
BAS (x10^3/µL)	0.023± 0.0166	0.021± 0.0088	0.020± 0.0112	0.043±+D 0.0134	0.0023±  0.0126	0.026± 0.0114
LUC (x10^3/µL)	0.019± 0.0166	0.020± 0.0141	0.024± 0.0113	0.054±+D 0.0158	0.020± 0.0082	0.068± 0.0554
EOSR (%)	1.40± 0.356	1.19± 0.281	0.94± 0.606	0.77± +C 0.231	1.73± 0.492	1.22± 0.487
LUCR (%)	0.33± 0.195	0.38± 0.132	0.42± 0.156	0.63± +D 0.134	0.45± 0.129	1.12± 1.047
*D Dunnett LSD Test Significant at the 0.05 level +D Dunnett LSD Test Significant at the 0.01 level *C Cochran and Cox Test Significant at the 0.05 level +C Cochran and Cox Test Significant at the 0.01 level   HGB: Haemoglobin HCT: Haematocrit LYMR/LYM: Lymphocytes (relative or absolute) MONR/MON: Monocytes (relative or absolute) EOSR/EOS: Eosinophils (relative or absolute) % or 10^3/µL BASR/BAS: Basophils (relative or absolute) % or 10^3/µL LUCR/LUC: Large unstained cells (relative or absolute)   Data shown as mean± Standard deviation

 

Table 4: Selected clinical chemistry data

Sex	Treatment Group (mg/kg bw/day)	Main study				Recovery phase
		Control	100	300	1000	Control	1000
	Number	10	10	9	10	4	5
M	ALP (U/L)	205.78± 42.116	201.75± 26.236	171.18± *D 23.076	165.61± *D 28.486	298.10± 58.376	235.40± 39.130
M	AST (U/L)	116.79± 17.488	96.17± *D 10.224	122.47± 21.731	95.40± *D 11.661	154.38± 37.481	155.14± 40.320
M	GLU (mg/dL)	156.33± 26.113	190.72± +C 14.039	142.73± 39.284	184.03± 36.541	138.18± 29.278	204.68± 62.246
F	GLU (mg/dL)	154.13± 45.932	144.36± 18.219	112.81± *C 20.487	118.29± *C 17.696	196.58± 19.242	198.70± 11.716
M	CREA (mg/dL)	0.379± 0.0431	0.387± 0.0662	0.365± 0.0310	0.329± +C 0.0213	0.313± 0.0222	0.346± 0.0385
M	Ca (mmol/L)	2.440± 0.0389	2.487± 0.0485	2.484± 0.0570	2.564±+D 0.0932	2.435±  0.0311	2.484± *D 0.0152
M	Na (mmol/L)	140.61± 1.680	141.16± 0.809	142.06± *C 0.747	140.82± 0.673	140.63± 0.310	139.80± 0.919
M	IP (mg/dl)	6.704± 0.6921	8.405± +C 1.3624	7.158± 0.8612	6.313± 0.2748	6.168± 0.7605	6.790± 0.2571
F	IP (mg/dl)	5.167± 0.6691	5.875± 0.9450	6.411± +D 0.6256	6.497± +D 1.1293	6.168± 0.5034	6.412± 0.8027
	*D Dunnett LSD Test Significant at the 0.05 level +D Dunnett LSD Test Significant at the 0.01 level *C Cochran and Cox Test Significant at the 0.05 level +C Cochran and Cox Test Significant at the 0.01 level   M = male, F = female ALP: Alkaline phosphatase AST: Aspartate aminotransferase GLU: Glucose CREA: Creatinine Ca: Calcium Na: Natrium IP:Inorganic phosphorus   Data shown as mean± Standard deviation

 

Table 5: Selected open field measurements

Sex	Day	Treatment Group (mg/kg bw/day)	Main study
			Control	100	300	1000
		Number	15	10	10	15
M	Allocation Day 5	RE	7.3± 1.05	6.0± +D 0.82	5.8± +D 0.92	6.0± +D 1.00
M	Dosing Day 5	RE	7.5± 2.61	6.1± 2.23	6.8± 2.10	6.2± 1.52
M	Dosing Day 13	RE	7.1± 2.37	6.2± 1.93	6.3± 3.09	6.7± 3.02
M	Dosing Day 20	RE	8.4± 1.24	8.4± 1.26	8.7± 1.16	6.9± +D 1.41
M	Dosing Day 27	RE	5.3± 1.68	3.2± +D 1.69	3.3± +D 1.77	4.8±  0.94
M	Dosing Day 34	RE	3.7± 1.23	4.2±  0.92	4.5±  0.85	4.3±  0.82
M	Dosing Day 41	RE	5.8± 1.53	5.5±  2.42	4.7±  1.06	6.0±  2.78
M	Dosing Day 48	RE	5.9± 2.53	5.0±  1.89	6.5±  1.58	5.4±  0.83
M	Dosing Day 55	RE	8.1± 2.95	7.1±  5.15	6.1±  1.52	4.3± #C  1.67
M	Dosing Day 62	RE	6.1± 1.41	6.3±  1.34	5.9±  0.99	6.8±  1.66
M	Dosing Day 69	RE	4.9± 1.38	5.2±  1.32	4.8±  1.32	5.6±  1.35
M	Dosing Day 76	RE	6.9± 2.11	6.9±  2.23	5.7±  1.64	7.5±  2.50
M	Dosing Day 83	RE	7.4± 1.50	6.7±  2.91	7.2±  2.70	7.0±  1.89
M	Dosing Day 90	RE	6.4± 1.55	7.2±  2.20	7.1±  1.79	4.7 *D±  1.23
F	Allocation Day 6	RE	14.4± 2.50	12.8± 2.49	8.9± +D 2.42	9.2± +D 1.21
F	Dosing Day 6	RE	14.1± 1.79	13.4± 2.12	12.9± 2.47	13.8± 2.04
F	Dosing Day 14	RE	13.2± 1.74	12.4± 2.59	12.1± 1.20	12.9± 2.28
F	Dosing Day 21	RE	14.9± 1.87	13.7± 2.71	15.9± 1.37	13.1± *D 2.02
F	Dosing Day 28	RE	7.8± 1.37	10.3± +D 1.42	9.1± 1.85	6.9± 1.96
F	Dosing Day 35	RE	7.1± 2.36	10.7± *C 4.08	7.5± 1.72	8.7± *C 1.23
F	Dosing Day 42	RE	13.1± 4.65	10.8± 4.18	10.1± 2.85	10.0± 2.04
F	Dosing Day 49	RE	10.2± 3.30	10.1± 3.87	10.5± 2.07	9.6± 2.47
F	Dosing Day 56	RE	10.3± 2.31	10.3± 1.95	9.6± 2.22	9.1± 1.92
F	Dosing Day 63	RE	11.2± 2.37	11.9± 2.13	10.9± 2.92	11.3± 2.43
F	Dosing Day 70	RE	8.7± 2.49	9.5± 2.46	10.4± 1.51	10.0± 2.11
F	Dosing Day 77	RE	8.7± 2.22	8.0± 2.71	10.0± 1.76	9.8± 2.22
F	Dosing Day 84	RE	10.9± 2.00	10.6± 2.01	11.4± 1.84	11.2± 1.31
F	Dosing Day 91	RE	10.5± 2.20	10.7± 2.54	11.1± 1.73	10.9± 1.82
*D Dunnett LSD Test Significant at the 0.05 level +D Dunnett LSD Test Significant at the 0.01 level *C Cochran and Cox Test Significant at the 0.05 level +C Cochran and Cox Test Significant at the 0.01 level RE: Rearing Data shown as mean± Standard deviation

 

Table 6: Selected Organ weight to terminal body weight (%) in males

Treatment Group (mg/kg bw/day)	Number	Testes	Adrenal gland
Control group	10	0.9274± 0.08141	0.0131± 0.00174
100	10	0.9407± 0.04834	0.0131± 0.00111
300	10	1.0133± 0.11447	0.0131± 0.00136
1000	10	1.0397± *D 0.07421	0.0146± *D 0.00124
Recovery phase
Control group	4	0.0111± 0.00034	0.9127± 0.03394
1000	5	0.0112± 0.00168	0.9293± 0.04947
*D =Dunnett LSD Test Significant at the 0.05 level Data shown as mean± Standard deviation

 

Table 7: Selected landing foot splay data

Sex	Treatment Group (mg/kg bw/day)	Main study				Recovery phase
		Control	100	300	1000	Control	1000
	Number	14	10	10	15	4	4
M	LAN 1 (cm)	5.64± 1.345	6.45± 1.066	6.95± *D 0.862	6.87± *D 1.316	6.88± 1.109	7.40± 1.517
M	LAM (cm)	5.661± 0.9383	6.725± 0.9534	6.752± 1.2343	6.719± *D 1.2607	7.375± 0.9242	7.450± 0.7786
	*D Dunnett LSD Test Significant at the 0.05 level +D Dunnett LSD Test Significant at the 0.01 level *C Cochran and Cox Test Significant at the 0.05 level +C Cochran and Cox Test Significant at the 0.01 level   LAN1: Landing foot splay 1 (first measurement of distance between ink blots (cm)) LAN2: Landing foot splay 2 (second measurement of distance between ink blots (cm)) LAM: Landing foot splay mean (averaged measurements of distance between ink blots (cm)) Data shown as mean± Standard deviation

 

Applicant's summary and conclusion

Conclusions:

No adverse effects were observed at all dose levels investigated following treatment with the test item, when administered by oral gavage for 13 consecutive weeks at the dosages of 100, 300 and 1000 mg/kg bw/day when compared to controls. One case of poor health conditions, which caused a premature sacrifice for humane reasons occurred in one female animal dosed at 1000 mg/kg bw/day. The relation to treatment with the test item of the illness status of this female cannot be established on the basis of macroscopic and microscopic findings. Based on these findings, it can be concluded that the NOAEL for this study is >=1000 mg/kg bw/day.