3-[4-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonamide, reaction products with aqueous organic chromium(III) complex sodium salt, then laked with acidified C10-14-tert-alkyl(linear and branched) amines

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-08-25 to 2016-04-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-[4-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonamide, reaction products with aqueous organic chromium(III) complex sodium salt, then laked with acidified C10-14-tert-alkyl(linear and branched) amines

EC Number:

939-383-2

Molecular formula:

UVCB substance

IUPAC Name:

3-[4-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonamide, reaction products with aqueous organic chromium(III) complex sodium salt, then laked with acidified C10-14-tert-alkyl(linear and branched) amines

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 002-122106

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

other: Wistar rats, strain Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11-13 weeks
- Weight at study initiation: 321.1 g – 355.1 g (males); 196.3 g – 222.2 g (females)
- Fasting period before study: No
- Housing: During the study period, the rats were housed individually in Polycarbonate cages type III supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²) and TECHNIPLAST, Hohenpeißenberg, Germany, with the following exceptions: During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III. Pregnant animals and their litters were housed together until PND (post natal day) 4.
- Diet: Ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: ad libitum
- Acclimation period: About 5 days

ENVIRONMENTAL CONDITIONS:
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air change: 15 times per hour
- Photoperiod:12 hours light (from 6.00 h to 18.00 h) and 12 hours darkness (from 18.00 h to 6.00 h)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test substance was weighed in a beaker depending on the test group and thoroughly mixed with a small amount of food (Ground Kliba maintenance diet mouse/rat). Then further amounts of food were added to this premix and thoroughly mixed. Afterwards, further amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 3 minutes in a laboratory mixer.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Maximum of 2 weeks
- Proof of pregnancy: Proof of pregnancy: Detection of mating was confirmed by sperm in vaginal smear. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1".
GD = gestation day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in the diet for a period of 35 days at room temperature were carried out before the study was initiated with a comparable batch. Samples of the test substance preparations were sent to the analytical laboratory once during the study period for verification of the concentration. The samples, which were taken for the concentration control analysis at the beginning of the administration period, were also used to verify the homogeneity.

Duration of treatment / exposure:

30 days (males);
50 – 56 days (females)

Frequency of treatment:

Daily

Details on study schedule:

- Age at mating of the mated animals in the study: 11 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 200 ppm

Remarks:

corresponding to 85 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

3 600 ppm

Remarks:

corresponding to 261 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

12 000 ppm

Remarks:

corresponding to 812 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

other: yes, treated with diet only

Details on study design:

- Dose selection rationale: Range-finder study

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Cage side observations included morbidity, pertinent behavioral changes and signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week. The follwoing exceptions are notable for the female animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14, and 20. Females with litter were weighed on the day after parturition (PND 1) and on PND 4.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testes weight, epididymides weight

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes. All pups were examined externally and eviscerated; their orangs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after 30 days
- Maternal animals: All surviving animals 50 and 56 days

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
Certain tissues (see "Any other information on materials and methods") were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on postnatal day (PND) 4.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including visceral findings.

Statistics:

Statistical analyses were performed with the following statistical tests:
Food consumption, body weight and body change, gestation days: DUNNET test (two-sided)
Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index, females with liveborn pups, females with all stillborn pups: FISHER'S EXACT test (one-sided)
Mating days until day 0 pc, % postimplantation loss, pups stillborn, % perinatal loss, implantation sites, pups delivered, pups liveborn, live pups day x, viability index: WILCOXON test (one-sided) with BONFERRONI-HOLM
% live male day x, % live female day x: WILCOXON test (two-sided)
Weight parameters (pathology): KRUSKAL-WALLIS test (two-sided) and WILCOXON-test (two-sided)

Reproductive indices:

Mating index (%), fertility index (%), gestation index (%), live birth index (%), postimplantation loss (%)

Offspring viability indices:

Viability index (%), sex ratio (%), stillborn and liveborn pups (%), postimplantation loss (%), perinatal loss (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
There were no test substance-related or spontaneous mortalities in any of the groups. All male and female F0 generation parental animals of test groups 1 to 3 (1200, 3600 and 12000 ppm) had dark red discolored feces throughout the entire study period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption of the high-dose F0 females was significantly below the concurrent control values during premating days 0-7 and 0-13 (about 14% and 9% less, respectively), during the entire gestation period (maximum of 15% less) and during the entire lactation period (about 31% less).
Further, a trend to a lower mean body weight gain was observed for the high-dose F0 females, and the difference to the control became significant on GD 20 (7.4% below control). During lactation period, mean body weights were significantly lower in female animals of test group 3 (14.3% below control on PND 4). Test group 3 females (12000 ppm) had a lower body weight gain during gestation (overall 18% below control) despite the difference was not statistically significant to the control. A significantly lower body weight gain was observed in female animals of test groups 2 and 3 (3600 and 12000 ppm) from PND 1 to PND 4 (about 46% and 67%, respectively). As the body weights in test group 2 were not different to control this single change in weight gain is not considered toxicologically relevant. The lower weight gain in test group 3 on the other hand was consistent with decreased body weights, therefore it is considered treatment-related and adverse.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The glandular stomach, jejunum and colon of almost all parental male and female animals of test groups 1 to 3 (1200, 3600 and 12000 ppm) showed red discoloration of the contents. This change was consistent with the red color of the test substance and was regarded as treatment-related but not adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

812 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Corresponds to 12000 ppm

2

Key result

Dose descriptor:

NOAEL

Remarks:

(fertility and reproductive performance)

Effect level:

812 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Corresponds to 12000 ppm

3

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

261 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Corresponds to 3600 ppm; reduced food consumption and impaired body weight development in females at 812 mg/kg bw/d (12000 ppm)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The viability index indicating pup mortality during lactation (PND 0-4) was 100%, 99.3%, 100% and 93.8% in test groups 0-3, respectively.

BODY WEIGHT (OFFSPRING)
Mean body weights of the high-dose (12000 ppm) male and female pups were significantly below the concurrent control values at PND 4 (average 24.6% below control). In addition, mean body weight change of the high-dose male and female pups was significantly below the concurrent control values from PND 1 to PND 4 (average 49%). The mean body weights and body weight change of the mid- and low-dose male and female pups were comparable to the concurrent control group throughout the entire study period. Four male and four female runts were recorded for test group 3 (12000 ppm) and one female runt was seen in the test group 2 (3600 ppm).

GROSS PATHOLOGY (OFFSPRING)
A few pups showed spontaneous findings at gross necropsy, such as dilated ureter, dilated renal pelvis, post mortem autolysis, empty stomach, and red discolored testis (left). These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

(developmental toxicity)

Generation:

F1

Effect level:

261 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Corresponds to 3600 ppm; Based on lower pup weights at 812 mg/kg bw/d (12000 ppm): Average pup weights 24.6 % below control; average weight gain 49 % below control

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Analysis 

The various analyses

- demonstrated the stability of the test substance preparations over a period of 35 days at room temperature

- confirmed the homogeneous distribution of the test substance in the diet

- verified correct concentrations of the test substance in the diet preparations
 
Absolute and relative weights

Absolute weights
When compared to control group 0 (set to 100%), the mean absolute terminal body weight of the parental females was significantly decreased as follows:

Absolut weights	Females
Test group (ppm)	1 (1200)	1 (3600)	3 (12000)
Terminal body weight	102 %	100%	94 %**

*: p<= 0.05, **: p <= 0.01

The findings in test group 3 were statistically significant.

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
All mean relative weight parameters did not show significant differences when compared to the control group 0.