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

Endpoint summary

Description of key information

key, Oral (OECD 408), rat: NOAEL ≥ 1000 mg/kg bw/day

key, Oral (OECD 422), rat: NOAEL ≥ 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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

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

Dose / conc.:

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

Dose / conc.:

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

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

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.

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.

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.

Critical effects observed:

no

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

 

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Jul 2012 - 16 Jan 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Erlangen, Germany

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 285-329 g (males), 173-209 g (females)
- Housing: single housing in IVC cages, type III H (except for mating period)
- Diet: Altromin 1324 maintenance diet for rats and mice (Lot No. 0939), ad libitum
- Water: tap water (sulphur acidified to pH 2.8), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 55±10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle (cottonseed oil) was added to give the appropriate final concentration of the test item. The formulation vials were placed on a Vortex machine for a short period to ensure proper homogenistation of the formulation. The test item formulation was prepared freshly on each administration day before the administration procedure. The time of preparation and time of dosing was recorded for all dosing formulations. The vehicle was also used as control item.


VEHICLE
- Justification for use and choice of vehicle: The vehicle has been selected on the basis of the test item’s characteristics.
- Concentration in vehicle (nominal): 25, 75, and 250 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Lot/batch no.: MKDJ0602V

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations. Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) from all groups (16 samples). The mean recoveries observed in low dose, middle dose and high dose groups were 102.5%, 101.4% and 101.9% of the nominal concentration. Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples). The mean recovery observed for the low dose group was 104.1 and 109.4% of nominal value, and 106.0 and 95.1% for high dose group. Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the preparation (at room temperature), from high and low dose formulations (4 samples). After 6 h storage at room temperature recovery compared to starting value was 88.0% and 100.6%.

Duration of treatment / exposure:

Males: at least 28 days (beginning during 14 days of pre-mating period)
Females: maximum period of 54 days (14 days pre-mating until post-natal day 3)

Frequency of treatment:

once daily (7 days per week)

Dose / conc.:

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

Dose / conc.:

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

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:
The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL. The doses were selected on the basis of data from a Dose Range Finding Study, where 3 animals per sex and dose (100, 300, and 1000 mg/kg bw/day) were treated in the same way as in the main study. No mortality and clinical findings were observed. Slightly reduced body weight development was observed in 300 and 1000 mg/kg bw dose groups. 1/3 animals of the high dose group did not deliver pubs and lost them during pregnancy. Based on these results 100, 300, and 1000 mg/kg bw were chosen as appropriate dose levels for the main study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded. Twice daily all animals were observed for morbidity and mortality. Females showing signs of abortion or premature delivery prior to the scheduled scarification of the animals were sacrificed and subjected to a thorough macroscopic examination.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and at least once a week thereafter.
- Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behavior were also recorded.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and on day 3 of the lactation period in 5 randomly selected females (only lactating females will be evaluated) outside the home cage using a functional observational battery of tests. Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE :
Food consumption was measured weekly on the respective days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

WATER CONSUMPTION: Not examined

HAEMATOLOGY/CLINICAL CHEMISTRY: Haematological parameters (differential blood count), clinical chemistry parameters and prothrombin time and activated partial thromboplastin time were examined in five males and five females randomly selected from each group at the end of the treatment period prior to or as part of the sacrifice of the animals. Clinical chemistry parameters included alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine, total protein (TP), albumin, urea, total bile acids (TBA), total cholesterol, glucose, sodium, and potassium.

URINALYSIS: An urinalysis was performed with samples collected from 5 randomly selected males prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded. Urinalysis parameters included specific gravity, nitrite, pH, protein, glucose, ketone bodies, urobilinogen, bilirubin, blood, leukocytes.

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals on study day 29 or 30 (28 day total dosing period)
- Maternal animals: All surviving animals on post natal day 4. Females showing no evidence of copulation after the mating period were sacrificed 24 to 26 days after the last day of the mating period.

GROSS NECROPSY
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved.

HISTOPATHOLOGY / ORGAN WEIGHTS
All full histopathology was carried out on the organs and tissues of 5 randomly selected males/females of the control and high dose group. These examinations were extended to animals of all other dosage groups for treatment-related changes that were observed in the high dose group. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) and all organs showing gross lesions were examined in all animals.
The following tissues were prepared for microscopic examination and weighed, respectively: brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), liver, uterus with cervix (females), kidneys, vagina (females), adrenal glands, testes (males), stomach, epididymides, (males, unilateral), small and large intestines (including Peyer´s patches), prostate and seminal, vesicles with coagulating glands as a whole (males), thymus, urinary bladder, thyroid, lymphnodes (mesentric and axillary), spleen, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, lungs and trachea, bone with bone marrow (sternum), mammary glands, pituitary gland, all gross lesions

Other examinations:

For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Statistics:

Comparison of dosed with control animals of the main groups was perfomed using one-way ANOVA and a post-hoc Dunnett Test for body weight, food consumption, parameters of haematology, blood coagulation, clinical biochemistry and absolute and relative organ weights. The statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

(piloerection and salivation through all dose groups (non-adverse))

Mortality:

mortality observed, treatment-related

Description (incidence):

(piloerection and salivation through all dose groups (non-adverse))

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased bw gain (day 1-7 of pre-mating period (high dose) and 0-4 during lactation (all test doses)) in females, non-adverse

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced food consumption (day 1-7, males), non-adverse

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

males: decreased spleen weight in high dose group, non-adverse; females: increased thyroid weight in low dose group, non-adverse

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinical symptoms were found in the control group. Slight piloerection was found in 5/10 high dose male animals (beginning on PMD 5), 3/10 medium dose male animals (beginning on MD3), and 1/10 low dose male animals (beginning on MD 14). Furthermore, 4/10 high dose female animals exhibited slight piloerection (beginning on PMD 7). Moving the bedding exhibited 3/10 male as well as 2/10 female high dose animals. Salivation was observed for 1/10 high and medium dose male animals as well as in 5/10 high dose and 1/10 low dose female animals. Besides these mentioned clinical symptoms alopecia at both forepaws and abnormal breathing were individually found in treatment groups.

BODY WEIGHT AND WEIGHT GAIN FOOD CONSUMPTION
In male animals, body weight increased with the progress of the study. No test item related influence on male body weight development or body weight gain could be detected. Food consumption was significantly decreased in the high dose group during pre-mating days 1-7.

In female animals, a statistically significant decreased body weight gain could be observed in the high dose group during pre-mating days 1-7 (3.6 compared to 9.9 g). Furthermore, during lactation days 0-4 a decrease in body weight was observed for treatment groups but not for control animals. However, this decrease in body weight was not dose dependent and not considered to be test item related. No change in food consumption could be measured for treatment groups when compared to the group.

HAEMATOLOGY
At the end of the treatment period all haematological parameters were within the normal range of variation. Furthermore, blood coagulation was not affected by test substance treatment.

CLINICAL CHEMISTRY
In male animals, values of ASAT were statistically significantly decreased in the low dose group when compared to control group (29.06 compared to 35.66). In the mid (31.32) and high (34.4) dose group, this decrease was slight and not statistically significant. Hence, this decrease is assumed to be not test item related and without toxicological relevance. All other parameters of clinical biochemistry were within the normal range of variation for this strain.

In female animals, values of TBA and ALP were increased in treatment groups when compared to controls. However, these increases were not dose-dependent and the variance within the individual groups was high. Hence, this increase cannot be clearly attributed to the test item and is assumed to be without toxicological relevance.
Besides, all parameters of clinical chemistry were within the normal range of variation for this strain and no clear difference could be observed when comparing treatment groups with control group.

URINALYSIS
The urinalysis performed in male animals revealed a slightly increased occurrence of blood in urine in high dose animals. However, neither histopathology nor organ weights nor macroscopic findings indicated that kidneys or urine bladder could be a target organ of the test substance An incidental occurrence of blood (e.g. due to contamination during the necropsy) seems likely. A toxicological relevance cannot be clearly stated. Furthermore, no test-item related effect in any of the treatment groups compared to the control group was observed for the other parameters of urine analysis.

NEUROBEHAVIOUR
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

ORGAN WEIGHTS
In male animals, absolute weights of spleens were significantly decreased in high dose group when compared to the control group. Relative spleen weights were slightly decreased in the high dose group which was not statistical significant. Since no toxicological relevant finding could was found during histopathological analysis, this weight decrease is considered to be not of toxicological relevance.

In female animals, relative thyroid/parathyroid weights were significantly increased in the low dose group (0.006±0.001 to 0.009±0.001). The same tendency could be found for absolute weights (0.018±0.003 g to 0.023±0.004 g). However, since no similar findings could be mentioned for the other test groups a test item relation cannot be stated.

GROSS PATHOLOGY
Few specific gross pathological changes were recorded for the male and female animals and were not considered to be treatment-related. Among others, yellow spots on epididymides (1/10 high dose males) or discoloured dark caecum (1/10 female control animals) were found.

HISTOPATHOLOGY: NON-NEOPLASTIC
No test item-related histopathological findings were noted in the reproductive organs and in the other organs evaluated in this study.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL corresponds to the highest dose tested.

Critical effects observed:

no

Table 1: Clinical observations of the male animals.

Clinical finding	Control	Low dose	Mid dose	High dose
Total number of animals examined	10	10	10	10
Slight piloerection	-	1	3	5
Moderate piloerection	-	-	-	1
Salivation	-	-	-	1
Severe salivation	-	-	1	-
Alopecia at both forepaws	-	1	1	-
Slight alopecia at both forepaws and at thigh	-	-	1	-
Moving the bedding	-	-	-	3

 Table 2: Clinical observations of the female animals.

Clinical finding	Control	Low dose	Mid dose	High dose
Total number of animals examined	10	10	10	10
Slight piloerection	-	-	-	4
Moderate piloerection	-	-	-	2
Piloerection	-	-	-	1
Slight alopecia at right forepaw	-	1	-	-
Alopecia at both forepaws	-	1	1	1
Salivation	-	1	-	5
Moderate salivation	-	1	-	-
Moving the bedding	-	-	1	5
Abnormal breathing	-	-	-	2

 

Table 3: Body weights (g) of the male animals.

	Day of study		Group
			Control	Low dose	Mid dose	High dose
Premating	P1	Mean	332.5	332.5	332.9	329.3
		SD	13.14	15.75	13.43	14.87
	P7	Mean	351.2	350.4	350.8	344.5
		SD	15.6	18.86	16.85	17.47
Mating and Post mating	MP1	Mean	366.1	365.6	366.6	357.1
		SD	16.83	18.86	20.91	19.25
	MP7	Mean	372.8	368.7	370.6	362.5
		SD	18.4	23.63	23.13	20.78
	MP14	Mean	380.6	377.6	376.3	372.8
		SD	17.71	23.32	21.62	18.02

Table 4: Body weights (g) of the female animals.

	Day of study		Group
			Control	Low dose	Mid dose	High dose
Premating	P1	Mean	204.8	203.7	204.1	206.1
		SD	9.72	10.2	10.32	9.72
	P7	Mean	214.7	212.8	212.5	209.7
		SD	10.98	10.11	14.71	13.74
	P14		221.7	222.3	216.8	216.5
			12.6	13.86	11.47	13.97
Gestation	GD0	Mean	223.67	222.67	221.44	218.33
		SD	9.99	12.76	20.0	12.58
	GD7	Mean	244.33	247.56	243.56	237.67
		SD	9.61	18.79	17.97	15.91
	GD14	Mean	274.33	277.44	271.22	266.67
		SD	8.91	19.18	19.05	17.87
	GD20	Mean	323.5	331.44	327.89	325.44
		SD	10.86	21.40	23.12	10.11
Lactation	L0	Mean	264.71	267.22	264.0	265.11
		SD	12.37	22.94	19.26	30.58
	L4	Mean	265.71	262.78	256.7	262.89
		SD	12.79	21.85	15.71	20.5

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid (DMPSA) was tested in a 13 week repeated dose oral toxicity study in rats with a 4 week recovery period according to OECD 408 and in compliance with GLP. Ten animals per dose and sex were treated per gavage with 100, 300, and 1000 mg/kg bw/day, respectively, 7 days per week in the main study.

Two cases of unscheduled death occurred during the study. One control male was sacrificed on Day 35, after swallowing the catheter. A high dose female wassacrificed on Day 48. Macroscopic and microscopic findings did notallow for a determination of the factors contributory to illness status of this female. Although a treatment-related effect cannot be entirely excluded, it is considered unlikely, since just one out 10 animals was affected.

Rales and/or decreased activity and/or ocular discharge were only observed in individual male animals dosed at 1000 mg/kg bw/day. Ocular discharge or a palpable mass were also seen in 2 males dosed at 100 mg/kg bw/day. No significant clinical signs were observed during the recovery period. No treatment-related clinical signs were observed in the females during treatment and recovery periods. Observation of animals at removal from the cage and in an open arena did not reveal changes of toxicological relevance.Thus, overall no relevant clinical signs were recorded during the study. In addition, no signs indicating neurotoxic effects were seen during the study. The slight statistically significant variations in mean rearing number recorded in treated males and/or females dosed at 100, 300 and 1000 mg/kg were not considered to be of toxicological relevance, due to the occasional occurrence of these change and in the absence of other signs. A slight statistically significant increase of mean landing footsplay was recorded in males treated at 1000 mg/kg bw/day (19%) at the end of treatment period. But no changes were observed at measurements performed at the end of recovery. Since these increases were not associated with other findings, they were not considered to be adverse.

Body weight, food consumption and oestrous cycle were not affected by treatment. Also, no lesions were recorded at ophthalmological examination.

Haematology analysis revealed an increase (58%) in white blood cells in high dose males, only. The observed changes were reversible during the recovery phase. No other treatment-related changes were observed. Since the effects were only observed in one sex and were reversible, they are not considered to be of toxicological relevance.

No treatment-related changes were recorded at the end of treatment andrecovery periods in coagulation and clinical chemistry parameters. The statistically significant 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. During the recovery phase, calcium was still slightly higher in males than in controls. As for the dosing phase, this change was considered to be incidental. The other statistically significant 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.

No treatment-relatedchanges were recorded for thyroid hormones levels.

At final sacrifice, an increase 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 significance in high dose males (+12%). Adrenal gland weight changes were not statistically significant. 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. Furthermore, at the end of the recovery period, there were no treatment-related changes 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.

There were no treatment-related microscopic observations in males and females receiving DMPSA up to and including 1000 mg/kg bw/day. There were also no treatment-related microscopic observations in the testes examined with PAS special stain. Any microscopic observations were within the range of expected spontaneous findings in SD rats of the same age considered incidental and unrelated to the test item.

In conclusion, no adverse effects on systemic toxicity after repeated exposure were observed. Therefore, a NOAEL of 1000 mg/kg bw/day was determined for systemic toxicity in the 90 day repeated dose toxicity study.

Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD 422 and in compliance with GLP in Crl:WI(Han) rats (Schleh, 2013). Ten animals per dose were treated per gavage with 100, 300, and 1000 mg/kg bw/d, respectively, 7 days per week. Male animals were treated for 28 days, beginning during the 14 days of the pre-mating period. Females were treated for a maximum period of 54 days, from 14 days of pre-mating period until post-natal day 4. The doses were selected based on data from a Dose Range Finding Study, where 3 animals per sex and dose (100, 300, and 1000 mg/kg bw) were treated in the same way as in the main study. No mortality and clinical findings was observed. Slightly reduced body weight gain was observed in the 300 and 1000 mg/kg bw/day dose groups. 1/3 animals of the high dose group did not deliver pubs and lost them during pregnancy. Based on these results 100, 300, and 1000 mg/kg bw/day were chosen as appropriate dose levels for the main study.

In the main study, no mortality was observed throughout the study period. Slight piloerection, moving the bedding and salivation were observed in various animals of the treatment groups. No test item related influence on male body weight development or body weight gain could be detected. Food consumption was significantly decreased in the high dose group during pre-mating days 1-7. In female animals, a statistically significant decreased body weight gain could be observed in the high dose group during pre-mating days 1-7. Furthermore, during lactation days 0-4 a decrease in body weight was observed for treatment groups but not for control animals. However, this decrease in body weight was not dose dependent and not considered to be test item related. No change in food consumption could be measured for treatment groups when compared to the control group. Slight decrease of spleen weights in males and slightly increased relative thyroid/parathyroid weights in females in individual dose groups were considered as toxicologically not relevant. No test item-related histopathological findings were noted in the organs evaluated in this study. No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.

In conclusion, no adverse effects on systemic toxicity after repeated exposure were observed. Therefore a NOAEL of ≥ 1000 mg/kg bw/day was deduced for systemic toxicity in the OECD 422 study.

Justification for classification or non-classification

The available data on the repeated dose toxicity of the test item do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.