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

Key value for chemical safety assessment

Effects on fertility

Description of key information

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

In accordance with Column 1 of REACH Annex IX the extended one-generation reproductive toxicity study does not need to be conducted as no adverse effects on reproductive organs or tissues were observed in the available OECD 422 screening study and the oral 90-day repeated dose toxicity study for the test substance DMPSA.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

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 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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- In case of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged: individually

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.

Parental animals: Observations and examinations:

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.
- 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 AND COMPOUND INTAKE : No data

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.

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

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.

Litter observations:

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing numbers on the back with the help of a permanent marker or by tattooing. In addition to the observations of parent animals, any abnormal behavior of the offspring was recorded.

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

SACRIFICE
- Dead pups and pups sacrificed on day 4 post-partum.

GROSS NECROPSY
- These animals were carefully examined externally for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Not examined

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).

Reproductive indices:

Number of corpora lutea and implantation sites, live pups (born on post-natal day 0) and pre- and post imlantation loss (%) were examined and calculated. In addition, the following indices were calculated:
Copulation Index (%) = (No. of rats copulated / No. of pairs) x 100
Fertility Index (%) = (No. of Females Pregant / No. of females copulated) x 100
Delivery Index (%) = (No. of dams with live newborns / No. of pregnant dams) x 100

Offspring viability indices:

Viability index (%) = (No. of live offspring at day 4/No. of live offspring at birth) X 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

piloerection and salivation in 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; reduced food consumption (day 1-7, males, high dose), non-adverse

Food consumption and compound intake (if feeding study):

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

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No clinical symptoms were found in the control group. Slight piloerection was found in 5/10 high dose male animals (beginning on pre-mating day (PMD) 5), 3/10 medium dose male animals (beginning on mating day (MD) 3), 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 FOOD CONSUMPTION (PARENTAL ANIMALS)
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.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No findings were reported after qualitative examination of testes, taking into account the tubular stages of the spermatogenic cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No influence of the test substance was noted regarding the reproductive performance of the parental animals. Three females of the control group, one female treated at 100 mg/kg bw/day and one female treated at 1000 mg/kg bw/day were found not to be pregnant at terminal sacrifice, but in view of the group distribution this was not considered to be treatment-related.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In male animals, absolute weights of spleens were significantly decreased in high doe 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 bewas found during histopathological analysis, this weight decrease is not 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 (PARENTAL ANIMALS)
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 of male animals (1/10 high dose animals) or discoloured dark caecum (1/10 female control animals) were found.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No test item-related histopathological findings were noted in the reproductive organs and in the other organs evaluated in this study.

FUNCTIONAL OBSERVATIONS (PARENTAL ANIMALS)
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.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse and treatment-related effects were observed up to and including the highest tested dose level.

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse and treatment-related effects were observed up to and including the highest tested dose level.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
No treatment-related effect on the litter data was observed for parameters such as the total number of pups born or live pups on PND 0 and PND 4. No still births or runts were recognized.

BODY WEIGHT (OFFSPRING)
No change in litter weight was observed in the treated groups.

GROSS PATHOLOGY (OFFSPRING)
No gross external findings were observed in any group.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse and treatment-related effects were observed up to and including the highest tested dose level.

Reproductive 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

  Table 5: Pre- and Post Natal Data and Mean Reproductive Indices.

		Group
		Control	Low dose	Mid dose	High dose
Corpora lutea	Mean	16.71	14.56	18.2	16.89
	SD	5.22	3.68	3.88	4.48
Implantation sites	Mean	9.86	11.22	11.7	11.67
	SD	2.61	2.59	1.06	1.5
Live pups born on PND 0		9.57	9.44	11.3	11.0
		2.57	2.24	1.34	2.06
% Pre Implantation loss	Mean	35.07	21.26	33.07	27.48
	SD	24.65	16.69	15.7	15.16
% Post Implantation loss	Mean	2.88	14.66	3.59	6.22
	SD	5.12	12.99	4.66	11.38
Viability Index (%)	Mean	100	100	100	100
	SD	0	0	0	0

 

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Effect on fertility: via oral route

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, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

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 guideline 422 and in compliance with GLP in Crl:WI(Han) rats (Schleh, 2013). Ten animals per dose were treated 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 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) 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. No influence of the test substance was noted regarding the reproductive performance of the parental animals. Three females of the control group, one female treated at 100 mg/kg bw/day and one female treated at 1000 mg/kg bw/day were found not to be pregnant at terminal sacrifice, but in view of the group distribution this was not considered to be treatment-related. In addition, no findings were reported after qualitative evaluation of testis, taking into account the tubular stages of the spermatogenic cycle. 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 reproductive organs and in the other 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.

No treatment-related effect on the litter data was observed for parameters such as the total number of pups born or live pups on post natal day 0 and 4. No still births or runts were recognized. No change in litter weight was observed in the treated groups, and no gross external findings were observed in any group.

In conclusion, no adverse effects on systemic or reproductive toxicity were observed. Therefore a NOAEL of ≥ 1000 mg/kg bw/day was deduced for systemic and reproductive toxicity in the OECD 422 study. No effects on developmental toxicity of the F1 generation were observed based on the available information, indicating a NOAEL of ≥ 1000 mg/kg bw/day for developmental toxicity.

Effects on developmental toxicity

Description of key information

Oral (OECD 414), rat: NOAEL ≥ 1000 mg/ kg bw/day

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

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Jun - 16 Jul 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

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
- Age at study initiation: 11 weeks (males), 7-9 weeks (females)
- Weight at study initiation: at least 313.3-352.2 g (males) 180.5-218.5 g (females)
- Housing: before and after mating, the animals were housed no more than 5 of one sex to a cage clear polysulfone cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese); nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week. During the mating period, one male rat was housed with one female rat in clear polysulfone cages measuring 42.5×26.6×18 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese) with a stainless steel mesh lid and floor.
- Diet: laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei 4, 20019 Settimo Milanese (MI), Italy), ad libitum
- Water: ad libitum
- Acclimation period: 4 weeks

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: 14 June 2021 To: 16 July 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of DMPSA was suspended in the vehicle using the following procedure:
– the required amount of test item was weighed
– the required amount of vehicle was added
– the mixture was shaken by hand
– the mixture was treated with a Silverson for 2-3 minutes
– the resulting suspension was left under magnetic stirring, at room temperature, for at least 1 hour prior to dosing or analysis.
The formulation was prepared daily from the start of the study up to June 28, thereafter weekly (concentrations of 25, 75, 250 mg/mL) up to July 12 and finally for three days, according to stability data obtained from ERBC Study No. A4260 and from the pre-formulation analysis performed during the course of this study. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle: corn oil, no justification provided
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle: 4 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 and it was extended up to 250 mg/mL in the present study. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85-115%; precision CV < 10%).
A 28 hour stability and an 8 day (9 day only for 250 mg/mL) stability at room temperature were verifed in the range from 20 to 250 mg/mL.
The proposed preparation procedure for the test item was checked in the range from 20 to 250 mg/mL by chemical analysis (concentration and homogeneity) in ERBC Study nos. A4260 and X1780 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 made during Week 1 and the last week of treatment 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).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight, until a positive identifcation of mating was made.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6 through Day 19 post coitum.

Frequency of treatment:

daily, 7 days/week

Duration of test:

20 days

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:

25

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: Animals were allocated to the groups by computerised stratifed randomisation to give approximately equal initial group mean body weights.
- Fasting period before blood sampling for rat dam thyroid hormones: not specified
- Time of day for rat dam blood sampling: in the morning of the day of necropsy

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, mortality was checked two times per day

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

FOOD CONSUMPTION : Yes
- Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: brain, thyroids

OTHER: Immunoanalysis - Thyroid hormone determination (T3, T4 and TSH) (delegated phase)
- Time schedule: on Day 20 post coitum

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.
- Other:
– Number, sex and weight of all live foetuses
– Number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing)
– Number of intra-uterine deaths
– Gross evaluation of placentae

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected: approximately 0.8 mL

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes

Statistics:

For continuous variables the signifcance of the differences amongst group means was assessed by analysis of variance (one-way Anova).
Inter-group differences were assessed by Dunnett’s test or a modifed t test, depending on the homogeneity of data (verifed by Bartlett’s test).
Statistical analysis of non-continuous variables was carried out by means of the KruskalWallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.

Indices:

Pre-implantation loss was calculated as a percentage from the formula:
Pre impl.Loss% = no.of corpora lutea−no.of implantations / no.of corpora lutea × 100

Post-implantation loss was calculated as a percentage from the formula:
Post impl.Loss% = no.of implantations −no.of live foetuses / no.of implantations × 100

Total implantation loss was calculated as a percentage from the formula:
Total impl.Loss% = no.of corpora lutea−no.of live foetuses / no.of corpora lutea × 100

Historical control data:

T3, T4, TSH Historical control data for Sprague Dawley rat in serum (2020-2021) was provided in tables (Please refer to "Any other information on materials and methods incl. tables" section).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

A scab was observed on the nose of a female from the control group on Day 20 post coitum.
On Day 6 of the gestation phase, tooth cut and salivation were observed each respectively in one mid-dose female and in one high dose animal.
These fndings were not considered related to the treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No animal died during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically signifcant reduction in body weight gain recorded on Day 9 post coitum in the high dose group compared to the control group was considered incidental since it was limited only on Day 9 post coitum. (Please refer to Table 1 in the "any other inforamtion on results incl. tables" section)

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No changes in food consumption were noted in all treated groups, when compared to controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

There was no effect on thyroid hormone determination.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related organ weight changes (brain and thyroid gland) at the end of the treatment period. Any variations were considered to be within the range of expected spontaneous changes in rats of the same age and unrelated to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic observations at the end of the treatment period. Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related microscopic observations in the thyroid gland at the end of the treatment period. Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There was no effect on thyroid hormone determination.

Number of abortions:

no effects observed

Description (incidence and severity):

(Please refer to Table 2 in the "any other information on results incl. tables" section)

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

(Please refer to Table 2 in the "any other information on results incl. tables" section)

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

(Please refer to Table 2 in the "any other information on results incl. tables" section)

Early or late resorptions:

no effects observed

Description (incidence and severity):

(Please refer to Table 2 in the "any other information on results incl. tables" section)

Dead fetuses:

no effects observed

Description (incidence and severity):

(Please refer to Table 2 in the "any other information on results incl. tables" section)

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The number of females with live foetuses on gestation Day 20 was 24 in each of the control, low and high dose groups and 25 in the mid-dose group.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

A total of 3 females were found not pregnant at necropsy: 1 each in the control, low and high dose groups. (non-treatment related and non adverse, no dose-response relationship, individual finding)

Other effects:

no effects observed

Description (incidence and severity):

Gravid uterus weight: No differences were detected in terminal body weight, uterus weight and absolute weight gain (terminal body weight minus gravid uterus weight, minus body weight at Day 0 post coitum) between control and treated groups.

Corpora lutea: no effects observed (Please refer to Table 2 in the "any other information on results incl. tables" section)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no treatment-related effects observed

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

(Please refer to Table 2 in the "any other information on results incl. tables" section)

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

(Please refer to Table 2 in the "any other information on results incl. tables" section)

Changes in sex ratio:

no effects observed

Description (incidence and severity):

(Please refer to Table 2 in the "any other information on results incl. tables" section)

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

(Please refer to Table 2 in the "any other information on results incl. tables" section)

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

No treatment-related differences were noted in the mean values of the anogenital distance of foetuses of both sexes maternally exposed at all dose levels compared to the control group. (Please refer to Table 2 in the "any other information on results incl. tables" section)

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A total of 6 foetuses were small (body weight below 2.7 g) at the external examination: 1 in the control group, 2 in the low dose group from 2 different litters, and 3 in the high dose group from 2 different litters.
Cleft palate was noted in 1 foetus of the control group. This fnding was confrmed as visceral malformation at the visceral examination of foetuses.
Anomalies, as hindlimb with abnormal shape and a rudimentary tail, were observed in one foetus of the control group and one foetus of the low dose group, respectively.
No other fndings were recorded in all groups. All observations are considered incidental, due to a missing dose-relationship and the occurence in the control group. (Please refer to Table 3 in the "any other information on results incl. tables" section)

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No malformation was observed at skeletal examination of foetuses. Anomalies and variations were noted both in control and treated groups with a similar incidence, suggesting no treatment-related adverse effect of the test item. (Please refer to Table 4 in the "any other information on results incl. tables" section)

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Cleft palate, observed at the external examination in 1 foetus of the control group, was confrmed at the foetal visceral examination.
Anomalies and variations were noted in control and treated groups with a similar incidence, suggesting no treatment-related adverse effect of the test item. (Please refer to Table 5 in the "any other information on results incl. tables" section)

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1: Body weight gain per day (g) of pregnant females – group mean

Dose group (mg/kg bw/day)	Number	Day of gestation
		3	6	9	12	15	18	20
Control	24	4.012± 1.337	3.227± 1.445	3.019± 1.344	4.298± 1.321	5.857± 1.636	13.584± 2.610	15.198± 3.274
100	24	4.339± 0.961	3.412± 0.944	2.970± 1.324	4.362± 0.965	5.388± 1.741	14.477± 1.777	15.140± 2.774
300	25	3.674± 2.666	2.960± 1.479	3.790± 2.940	4.297± 1.385	6.038± 1.745	13.639± 2.662	14.528± 3.574
1000	24	3.806± 1.093	3.509± 1.120	2.110*± 1.552	4.925± 1.557	6.019± 1.562	14.427± 2.683	15.432± 2.566
* = mean value of group is significantly different from control at p < 0.05 Data shown as mean± standard deviation

 

Table 2: Litter data and sex ratios – group mean

Dose group (mg/kg bw/day)	Control	100	300	1000
Number	24	24	25	24
Corpora lutea	15.17± 2.12	13.92± 2.54	14.36± 3.32	14.33± 2.06
Implantations	14.25± 2.19	13.33± 2.04	13.56± 3.27	13.79± 1.98
Uterine deaths early	0.63± 2.24	0.08± 0.28	0.28± 0.61	0.29± 0.75
Uterine deaths late	0.04± 0.20	0.04± 0.20	0.00± 0.00	0.08± 0.41
Uterine deaths total	0.67± 2.26	0.13± 0.34	0.28± 0.61	0.38± 0.82
Viable total	13.58± 3.05	13.21± 2.00	13.28± 3.39	13.42± 2.08
Viable males	6.50± 1.82	6.67± 2.30	6.96± 2.61	6.75± 1.94
Viable females	7.08± 2.19	6.54± 1.98	6.32± 2.43	6.67± 2.14
% males	48.19± 9.33	49.89± 15.03	52.37± 13.46	50.58± 13.70
Pre-Implantation loss %	6.05± 7.55	3.54± 5.85	5.58± 8.51	3.63± 6.04
Post-Implantation loss %	4.56± 15.10	0.88± 2.38	2.31± 5.23	2.74± 5.70
Implantation loss % total	10.55± 15.43	4.40± 6.06	7.71± 10.14	6.28± 7.86
Litter weigth (g)	49.38± 11.02	48.02± 7.27	48.25± 11.73	50.33± 8.24
Mean foetal weight males	3.76± 0.23	3.77± 0.17	3.74± 0.22	3.84± 0.19
Mean foetal weight females	3.56± 0.21	3.51± 0.19	3.57± 0.25	3.66± 0.22
Weight combined (g)	3.65±0.21	3.64±1.67	3.66±0.23	3.75± 0.19
* = mean value of group is significantly different from control Data shown as mean± standard deviation

 

Table 3: External examination of fetuses

Dose group (mg/kg bw/day)	Organ, Category, Observation	Number of fetuses affected	%
Control	Forelimb AN Abnormal shape	1	0.31
	Head MA Cleft palate	1	0.31
	Whole foetus No abnormalities detected	323	99.08
	Whole foetus AN Small	1	0.31
100	Tail AN Rudimentary	1	0.32
	Whole foetus No abnormalities detected	314	99.05
	Whole foetus AN Small	2	0.63
300	Whole foetus No abnormalities detected	332	100.00
1000	Whole foetus No abnormalities detected	319	99.07
	Whole foetus AN Small	3	3
VA = Variation, AN = Anomaly, MA = Malformation

 

Table 4: Skeletal examination of fetuses

Dose group (mg/kg bw/day)	Organ, Category, Observation	Number of fetuses affected	%
Control	Forepaw(s) AN Metacarpal(s) no ossification	34	20.24
	Ribs VA Rudimentary	4	2.38
	Ribs VA Short	14	8.33
	Ribs VA 14 ribs	2	1.19
	Skull VA Interparietal incomplete ossification	8	4.76
	Skull VA Supraoccipital incomplete ossification	44	26.19
	Sternebrae AN No ossification	2	1.19
	Sternebrae AN Asymmetrical ossification	5	2.98
	Sternebrae VA Incomplete ossification	32	19.05
	Sternebrae VA No ossification	7	4.17
	Sternebrae VA Incomplete ossification	16	9.52
	Thoracic vertebrae AN Centrum bipartite	2	1.19
	Thoracic vertebrae VA Centrum incomplete ossification	7	4.17
	Thoracic vertebrae VA Centrum dumb-bell shaped	2	1.19
100	Forepaw(s) AN Metacarpal(s) no ossification 4th	29	17.68
	Ribs VA Rudimentary 14th	4	2.44
	Ribs VA Short 14th	12	7.32
	Ribs VA 14 ribs	1	0.61
	Sacral vertebrae AN Arch(es) incomplete ossification	1	0.61
	Skull VA Interparietal incomplete ossification	7	4.27
	Skull VA Supraoccipital incomplete ossification	34	20.73
	Sternebrae AN Bipartite 5th	1	0.61
	Sternebrae AN Asymmetrical ossification	4	2.44
	Sternebrae AN Asymmetrical ossification 5th	2	1.22
	Sternebrae AN No ossification	1	0.61
	Sternebrae VA Incomplete ossification 5th	22	13.41
	Sternebrae VA Incomplete ossification 6th	33	20.12
	Sternebrae VA No ossification 5th	3	1.83
	Sternebrae VA Incomplete ossification	1	0.61
	Thoracic vertebrae AN Centrum bipartite	1	0.61
	Thoracic vertebrae VA Centrum dumb-bell shaped	5	3.05
	Thoracic vertebrae VA Centrum incomplete ossification	4	2.44
300	Forepaw(s) AN Metacarpal(s) no ossification 4th	31	17.82
	Ribs VA Rudimentary 14th	4	2.30
	Ribs VA Short 14th	13	7.47
	Skull VA Interparietal incomplete ossification	3	1.72
	Skull VA Supraoccipital incomplete ossification	43	24.71
	Sternebrae AN Asymmetrical ossification 5th	2	1.15
	Sternebrae AN Asymmetrical ossification	3	1.72
	Sternebrae VA No ossification 5th	3	1.72
	Sternebrae VA Incomplete ossification 6th	34	19.54
	Sternebrae VA Incomplete ossification 5th	26	14.94
	Thoracic vertebrae VA Centrum dumb-bell shaped	1	0.57
	Thoracic vertebrae VA Centrum incomplete ossification	8	4.60
1000	Forepaw(s) AN Metacarpal(s) no oss. 4th	25	14.97
	Lumbar vertebrae AN Centrum bipartite and asymmetrical ossification	1	0.60
	Ribs VA Rudimentary 14th	6	3.59
	Ribs VA Short 14th	15	8.98
	Skull VA Supraoccipital incomplete ossification	26	15.57
	Skull VA Interparietal incomplete ossification	5	2.99
	Sternebrae AN Asymmetrical ossification	5	2.99
	Sternebrae AN No ossification 6th	1	0.60
	Sternebrae AN Asymmetrical ossification 5th	3	1.80
	Sternebrae VA Incomplete ossification	2	1.20
	Sternebrae VA Incomplete ossification 5th	23	13.77
	Sternebrae VA Incomplete ossification 6th	17	10.18
	Sternebrae VA No ossification 5th	1	0.60
	Thoracic vertebrae AN Centrum bipartite and asymmetrical	2	1.20
	Thoracic vertebrae AN Centrum bipartite	2	1.20
	Thoracic vertebrae VA Centrum incomplete ossification	10	5.99
	Thoracic vertebrae VA Centrum dumb-bell shaped	2	1.20
VA = Variation, AN = Anomaly, MA = Malformation

 

Table 5: Visceral Examination of foetuses

Dose group (mg/kg bw/day)	Organ, Category, Observation	Number of fetuses affected	%
Control	Abdomen VA Haemorrhagic	5	3.16
	Abdomen VA Umbilical vein left sided	2	1,27
	Great vessels VA Innominate artery short	1	0.63
	Head MA Cleft palate	1	0.63
	Heart AN Atrium enlarged slight	2	1.27
	Kidneys AN Ectopic	1	0.63
	Kidneys VA Pelvic dilatation slight	3	1.90
	Testis AN Displaced	10	6.33
	Ureter VA Enlarged slight	13	8.23
100	Abdomen VA Umbilical vein left sided	3	1.96
	Kidneys AN Pelvic dilatation moderate	2	1.31
	Kidneys VA Pelvic dilatation slight	1	0.65
	Testis AN Displaced	10	6.54
	Ureter AN Enlarged moderate	3	1.96
	Ureter VA Enlarged slight	8	5.23
300	Abdomen VA Umbilical vein left sided	4	2.56
	Abdomen VA Haemorrhagic	5	3.21
	Heart AN Atrium enlarged slight	1	0.64
	Kidneys VA Pelvic dilatation slight	1	0.64
	Testis AN Displaced	4	2.56
	Ureter VA Enlarged slight	5	3.21
1000	Abdomen VA Umbilical vein left sided	1	0.65
	Abdomen VA Haemorrhagic	5	3.25
	Heart AN Atrium enlarged slight	4	2.60
	Kidneys AN Ectopic	1	0.65
	Kidneys VA Pelvic dilatation slight	1	0.65
	Testis AN Displaced	11	7.14
	Ureter VA Enlarged slight	5	3.25
VA = Variation, AN = Anomaly, MA = Malformation

 

Conclusions:

Based on the results obtained in this prenatal developmental toxicity study in rats, the NOAEL for maternal and developmental toxicity can be set at 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

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 IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

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 an oral prenatal developmental toxicity study in SD rats from Day 6 to Day 19 post coitum, according to OECD 414 and in compliance with GLP .

Females were mated with males of the same strain and then assigned to 4 groups of 25 females each. DMPSA dissolved in corn oil was tested at 100, 300 and 1000 mg/kg bw/day. Control animals receivedthe vehicle (corn oil) during the same treatment period. Dose levels were selected, based on the results obtained in a previous oral toxicity study in rats (OECD 422) and a preliminary non-GLP study, in which no adverse effects were noted at all dose levels (100, 300 and 1000 mg/kg bw/day).

Mortality, body weight, clinical signs and food consumption were recorded during thein vivophase. On Day 20post coitumallfemales were caesarian sectionedand subjected topost mortemexaminations. Blood was collected for hormone determination (T3, T4, TSH). Furthermore, brain and thyroid weights were analysed.

The number of pregnant rats,corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, sex, foetal and litter weight were recorded. Also the anogenital distance (AGD) was recorded. All foetuses were examined for external abnormalities. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities in all groups.

No animal died during the study and there were no-treatment related clinical signs observed in any dose-group. A statistically reduction in body weight gain recorded on Day 9post coitumin the high dose group compared to the control group was considered incidental since it was limited only to that day. There were no changes in food consumption. Thyroid hormone determination did not show any effect between treated and control animals. In line with this finding, there were no treatment-related organ weight changes (brain and thyroid gland) at the end of the treatment period.

No treatment-related macroscopic and microscopic (thyroid) observations were noted. There were also no maternal toxic effects observed at any dose level. A total of 3 females were found not pregnant at necropsy: 1 each in the control, low and high dose groups. This finding was considered incidental and not treatment-related. The number of females with live foetuses on gestation Day 20 was 24 in each of the control, low and high dose groups and 25 in the mid-dose group and thus unaffected by treatment. In addition, no differences were detected in terminal body weight, uterus weight and absolute weight gain (terminal body weight minus gravid uterus weight, minus body weight at Day 0post coitum) between control and treated groups. The number of corpora lutea, implantations, viable foetuses and intrauterine deaths were comparable between control and treated groups. There was no indication for foetal developmental toxicity. No treatment-related effects were seen in the mean foetal and litter weight, number of live offspring and changes in sex ratio. The external examination of fetuses, including the measurements of the anogenital distance, did not show differences in treated animals at all dose levels, when compared to the control group. No treatment-related findings were noted at skeletal and visceral examinations of foetuses.

Based on the results obtained in this study, the NOAEL for maternal and developmental toxicity was set at 1000 mg/kg bw/day.

 

 

In a study according to OECD 422 no effects on development have been observed with Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid. For further details refer to the discussion section "Effects on fertility".

Justification for classification or non-classification

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

No data are available regarding effects via lactation.