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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 13 July 1990 to 20 June 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted : 12 May 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methyl-1-phenyl-5-pyrazolone
EC Number:
201-891-0
EC Name:
3-methyl-1-phenyl-5-pyrazolone
Cas Number:
89-25-8
Molecular formula:
C10H10N2O
IUPAC Name:
5-methyl-2-phenyl-2,3-dihydro-1H-pyrazol-3-one
Test material form:
solid: particulate/powder
Remarks:
white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 8060110
- Expiration date of the lot/batch: not specified
- Purity test date: 12 December 1989

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: smocked glass bottle , stored at room temperature
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: Chemical analysis of preparation was performed by the Sponsor, samples of suspensions were taken as follows :
homogeneity : before the beginning of the study, the exact measurement of 2 x 10 ml, was taken at 3 different levels of the suspension of the highest concentration and frozen.
concentration : the exact measurement of 2 x 10 ml of each preparation, control group included, was taken on the first day of treatment and frozen.
These samples were sent in order to perform chemical analysis. The results of these analyses were not included in the report

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was suspended in the vehicle and homogeneized by a magnetic stirrer
- Final dilution of a dissolved solid, stock liquid or gel: not specified
- Final preparation of a solid: dissolved in vehicle

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl CD (SD) BR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (France)
- Age at study initiation: 10-12 weeks old
- Weight at study initiation: 230g
- Fasting period before study: not specified
- Housing: individually housed in U.A.R. cages (48 x 27 x 20cm)
- Diet (e.g. ad libitum): free access to the food U.A.R. A04C (Batch no. 00606, 00622, 00823 and 01017)
- Water (e.g. ad libitum): drinking water filtered using Millipore filters 0.22 micron)
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 50±20%
- Air changes (per hr): approximately 13 cycles / hour of filtered, non recycled air
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: First arrival (supplied to obtain 100 mated females): From: 19 september 1990 To: 1 November 1990
Second arrival (supplied to obtain 34 mated females) From : 6 November 1990 To: 12 December 1990

Animal numbers and assignements were presented in table 1 below.

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% carboxymethylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in the vehicle and homogenized by a magnetic stirrer. The preparations were performed daily and delivered protected from light by aluminium foil.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified, according to the instruction of the sponsor
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): not specified
- Lot/batch no. (if required): water provided bt Biosedra (France) Batch No. 1308, carboxymethyl cellulose provided by Prolabo (France) batch No. 88314
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis of preparation was performed by the Sponsor, samples of suspensions were taken as follows :
homogeneity : before the beginning of the study, the exact measurement of 2 x 10 ml, was taken at 3 different levels of the suspension of the highest concentration and frozen.
concentration : the exact measurement of 2 x 10 ml of each preparation, control group included, was taken on the first day of treatment and frozen.
These samples were sent in order to perform chemical analysis. The results of these analyses were not included in the report
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male / 2 females
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no
- 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:
The test substance was administered from day 6 to day 15 of pregnancy
Frequency of treatment:
The test substance was administered daily, 7 days per week
Duration of test:
until the day 20 of pregnancy
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 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:
32 to 35 females per test group and control
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were chosen according to a preliminary segment II study performed at the 100, 300, 1000 mg/kg/day dose levels (CIT/Study no. 6113 TSR) The results did not show maternotoxicity or embryotoxicity at any dose level. Therefore, the 1000 mg/kg/day dose level was chosen as the high dose level in the main study. This dose represents a very high safety factor when considering the human exposure level. The 40 mg/kg/day dose level was chosen as the low dose and the 200 mg/kg/day was the intermediate dose.

- Rationale for animal assignment (if not random): randomly
- Other: The concentration of these preparations were calculated so that each animal received a volume of 5 ml/kg/day. The volume of the daily administration was adjusted to the most recent bodyweight.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observed daily

BODY WEIGHT: Yes
- Time schedule for examinations: at day 0, 6, 9, 12, 15 and 20 of the pregnancy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
The measurement of food consumption was performed during pregnancy at the intervals day 0- day 6, day 6 - day 9, day 9 - day 12, day 12 - day 15 and day 15 - day 20
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The ovaries and the uterine horns
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of live and dead fetuses
Fetal examinations:
- External examinations: Yes: for all per litter
- Soft tissue examinations: Yes: for the first 20 litters obtained in each group
- Skeletal examinations: Yes: for the first 20 litters obtained in each group
- Head examinations: No
Statistics:
All means were accompanied by standart-deviations (S.D.) Comparison of bodyweight, food consumption, litter data (numbers of corpora lutea, implantation sites, fetuses, resorptions, fetal body weight) were performed by Dunnett's test. The proportions of live fetuses, post-implantation loss, and fetuses with abnormalitites were compared with Fisher exact test. All statistical analyses compared the treated groups to the control group with levels of signicance at p<0.05, p<0.01 and p<0.001. The tests were performed on a Repro/Toxicology system software.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the 1000 mg/kg/day group, all the treated animals presented orange coloured bedding from day 7 to day 16, except one female whose bedding was normal on day 16. Hypersalivation was observed in one female from day 15 to day 20
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 1000 mg/kg/day , a slight body weight loss was observed from day 6 to day 9. After day 9, the body gain was lower than that of the control group during the day 9 - day 15 period. From day 15 to day 20, the body weight gain was similar to that of the control group. Thus, the body weight gain during treatment period was lower than that of control group, corresponding to 15% and 5% of body weight gain for the control and the 1000 mg/kg/day respectively.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 1000 mg/kg/day group, it was slighty lower than that of the control animals, from day 6 to day 9, days 9 to 12, days 12 - 15 and days15 - 20. Thus, from days 6 to 20, the mean difference from the control group was about 19%.
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
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 1000 mg/kg/day, the body weight was lower than that of the control group
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
On the 40 mg/kg/day group, 7 fetuses was observed of the same litter presented a twisted tail. As this malformation was observed only in 1/22 liiters and was not found at highest dose levels, it was considered as incidental. In the 1000 mg/kg/day group, one fetus presented an umbilical hernia. Due to the very low frequency, this malformation was considered as incidental.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal variations : In the 1000 mg/kg/day group, the rate of unossified 5th sternebra was higher than that of the control group (67.5 vs. 34%;^p<0.001%). As a lower fetal body weight was observed in this group, this delayed ossification was related to the decrease of fetal body weight.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the 1000 mg/kg/day group, one out of 149 fetuses presented a pelvic renal ectopy (same fetus than that presented an umbilical hernia)

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in litter size and weights
other: delayed ossification

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

MEAN BODY WIEGHT OF FEMALES AND FETUSES

 

Dose

0

100

300

1000

DAY 0

MEAN

272 d

267

277

277

S.D.

23

23

17

3

N

23

22

17

29

DAY 6

MEAN

316 d

315

325

322

S.D.

22

19

18

26

N

23

22

22

24

DAY 9

MEAN

348

350

353

328

S.D.

24

19

20

25

N

23

22

22

24

DAY 12

MEAN

348 d

350

353

328 *

S.D.

23

23

18

24

N

23

22

22

24

DAY 15

MEAN

364 d

366

369

339 **

S.D.

25

25

18

23

N

23

22

22

24

DAY 20

MEAN

434 d

431

439

410

S.D.

44

45

31

32

N

23

22

22

24

Fetalbodyweight(g)

MEAN

3.74 d

3.74

3.63

3.43*

S.D.

0.45

0.47

0.26

0.30

 d=ANOVA + Dunnett's test

SUMMARY OF FETAL SKELETAL FORMATIONS

 

Dose

0

100

300

1000

5th sternebre unossified

Fetal incidence

N

50 f

46

49

104#

%

34.0

34.1

32.5

67.5

6th sternebre unossified

Fetal incidence

N

28 f

46 **

42

29

%

19.0

34.1

27.8

18.8

5th sternebre reduced ossification

Fetal incidence

N

58 f

61

74

28 #

%

39.5

45.2

49.0

18.2

 ** = p<0.01 #<= p<0.001

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the study, the registered substance 1-phenyl-3-methyl-5-pyrazolone did not induce developmental toxicity as teratogenicity to females rats after test item exposure by oral route during day 6 to 15 of pregnancy. Howerver, at the high dose level (1000 mg/kg/day), the registered substance induced clinical signs as orange colouration of bedding and a diminution of bodyweight. At 1000 mg/kg/day, maternal toxic effects were observed and associated with lower foetal weight and increased incidence of foetuses with reduced ossification. There were no teratogenic effects, and the NOEL was defined as 200 mg/kg/day for both maternal and developmental toxicity according to REACh regulation.
Executive summary:

This GLP-compliant study was performed to assess the potential teratogenicity of the registered item by developmental toxicity test, according to OCED Guideline 414 method ( teratogenicity, adopted 12 May 1981).

Three females Sprague-Dawley rats groups (32 to 35 females per group) and one vehicle control group were used, dose-levels selected were : 20, 200 and 1000 mg/kg/day. Females were mated with males. After positive mating, at day 6 to 15 days of pregnancy, animals were treated with test item by oral route. They were sacrified at day 20. Clinical examinations, bodyweight were oberserved. Maternal necropsy, litter parameters and fetal external observations were performed too.

Under the experimental conditions of the study, the registered substance 1-phenyl-3-methyl-5-pyrazolone did not induce developmental toxicity as teratogenicity to females rats after test item exposure by oral route during day 6 to 15 of pregnancy. Howerver, at the high dose level (1000 mg/kg/day), the registered substance induced clinical signs as orange colouration of bedding and a diminution of bodyweight. At 1000 mg/kg/day, maternal toxic effects were observed and associated with lower foetal weight and increased incidence of foetuses with reduced ossification. There were no teratogenic effects, and the NOEL was defined as 200 mg/kg/day for both maternal and developmental toxicity according to REACh regulation.