Registration Dossier

Administrative data

Description of key information

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
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:
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. certificate)
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 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:
subacute
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 VIII, 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 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.