Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 421
Version / remarks:
1995-07-27
Deviations:
no
GLP compliance:
yes
Remarks:
Research Toxicology Center S.p.A., Pomezia, Italy
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Identifier: CAS 80675-49-6
- Lot/batch No.: L-7015-20
- Yellow powder
- Storage conditions: Ambient temperature, no protection from light necessary

Test animals

Species:
rat
Strain:
other: HsdBrl: wistar Han rats
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l.
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: (P) ♂: 183 - 197 g; ♀: 155 - 164 g
- Housing: in a limited access rodent facility, pre-mating period: 5 animals per sex per cage, mating period: one male to one female, after mating: animals were housed individually
- Diet: commercially available laboratory powdered rodent diet (4 RF 21, Mucedola R.r.l., Italy)
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 25
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): fresh diet were prepared according to the stability of the test item in the diet.
- Mixing appropriate amounts with (Type of food): commercially available laboratory powdered rodent diet (4 RF 21, Mucedola R.r.l., Italy)
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed to confirm that the proposed formulation procedure is acceptable and that the homogeneity and stability of the formulation are satisfactory. Samples of the formulations prepared in week 1 and last week of the study were analysed to check the homogeneity and concentration. Chemical analysis was carried out by the Analytical Chemistry Department at RTC. The software used for this activity was the Empower® Pro build No. 2154.
Details on mating procedure:
- M/F ratio per cage: monogamous
- Length of cohabitation: until mating
- Proof of pregnancy: vaginal plug or sperm in vaginal smear was referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged in individual clear polycarbonate cages with stainless mesh lid and floor.
- Any other deviations from standard protocol: - for females from day 14 to day 20 post coitum and from day 0 to day 4 post partum the achieved dosage was calculated at 6 and 4 days interval respectively and not weekly as indicated in the study protocol.
Duration of treatment / exposure:
♂: 7 days a week for 2 consecutive weeks prior to pairing and thereafter until day of necropsy (total of 30-31 days of treatment).
♀: 7 days a week for 2 consecutive weeks prior to pairing and thereafter during mating, gestation and post partum until day 4 post partum.
Frequency of treatment:
daily, 7 days a week
Duration of test:
♀: 54 days, ♂: 30 - 31 days
Doses / concentrationsopen allclose all
Dose / conc.:
1 500 ppm (nominal)
Dose / conc.:
5 000 ppm (nominal)
Dose / conc.:
15 000 ppm (nominal)
Dose / conc.:
123 mg/kg bw/day (nominal)
Remarks:
♂: mean dose value as calculated from the reported body weight and standard food intake values.
Dose / conc.:
420 mg/kg bw/day (nominal)
Remarks:
♂: mean dose value as calculated from the reported body weight and standard food intake values.
Dose / conc.:
1 271 mg/kg bw/day (nominal)
Remarks:
♂: mean dose value as calculated from the reported body weight and standard food intake values.
Dose / conc.:
151 mg/kg bw/day (nominal)
Remarks:
♀: mean dose value as calculated from the reported body weight and standard food intake values.
Dose / conc.:
537 mg/kg bw/day (nominal)
Remarks:
♀: mean dose value as calculated from the reported body weight and standard food intake values.
Dose / conc.:
1 496 mg/kg bw/day (nominal)
Remarks:
♀: mean dose value as calculated from the reported body weight and standard food intake values.
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: dose levels were selected in consultation with the sponsor.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment groups. Males additionally on day that treatment commenced, weekly thereafter and just prior to necropsy. Females: weekly from the first day of treatment to mating, on days 0, 7, 14, and 20 post coitum and on days 1 and 4 post partum.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: males after mating, females & pubs 4 days post partum
- Organs examined: see Table 1
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 resorptions: Yes
Fetal examinations:
- External examinations: Yes, all per litter
- sex ratio, mortality, weights
Statistics:
For continous variables significance of differences amongst group means were assessed by Dunnett's test or a modified t-test depending on homogeneity of data. Non-continous variables were statistically analyzed by means of Kruskal-Wallis Test and intergroup differences between the control and treated groups assessed by a non-parametric version of Williams test. Statistical analyzes of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test. The mean values and standard deviations were calculated from actual values in the computer without rounding off.
Indices:
♂:
Copulatory Index (%) = No. of animals mated x 100/No. of animals paired
Fertility Index (%) = No. of males which induced pregnancy x 100/No. of males paired

♀:
Copulatory Index (%) = No. of animals mated x 100/No. of animals paired
Fertility Index (%) = No. of pregnant females x 100/No. of females paired

♂ and ♀:
Copulatory Interval = Mean number of days between pairing and mating

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
Effects seen were not treatment related. One female in the control group, 2 in the low dose group, one in the mid dose group and one in the high dose group were not pregnant. Unilateral implantation was present in one mid-dose and one high-dose female. One mid-dose female had unilateral total resorption and one high dose female had total litter loss. The number of dams with live pups on day4 post partum was 9 in the control group and 8 in the treated groups, respectively. The incidence of pregnancy outcome is comparable with the historical control data (of segment II studies).

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
15 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 496 mg/kg bw/day
Based on:
other: mean dose value as calculated from the reported body weight and standard food intake values
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
No treatment related effects were seen (for results see tables in "Any other information on results incl. tables).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
> 15 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 2: Implantation, pre-birth loss and gestation length - Group mean data

 Group    Corpora Lutea  Implantations  Total Litter Size  Pre-birth loss (%)  Gestation length (days)
 1 Mean 13.22 13.00 11.89 8.70 22.0
  Std  1.56  1.58 1.83 7.40 0.71
  9 9 9 9 9
2 Mean 11.50  11.00 9.38 16.95  22.0
   Std  3.25  3.96  3.54  11.9  0.53
   N  8  8  8  8  8
 3  Mean  12.25  12.00  11.25  7.29  22.00
   Std 3.15   3.07  3.15  6.23  0.00
 N  8  8  8  8  8
 4  Mean  12.50  11.00  9.50  14.90  22.0
   Std  2.83  4.00  4.31  20.31  0.53
   N  8  8  8  8  8

Table 3: Litter data at birth, on day 1 and on day 4 post partum - Group mean data

     at birth              on day1 post partum        on day4 post partum   

 Group  
   Total litter size  Live litter size  Pup loss [%]  Litter weight (g)  Mean pup weight (g)  Live litter size  Cumulative loss (%)  Litter weight (%) Mean pup weight (g)   
 1 Mean   11.89 11.89 0.00  75.71   6.36  10.33  14.09  90.11  8 .29  
   Std  1.83  1.83  0.00  14.45  0.68  3.24  22.38  38.04  1.95  
   N  9  9  9  9  9  9  9  9  9  
 2  Mean  9.38  9.13  2.39  60.65  6.88  8.63  7.44  83.03  9.84  
   Std  3.54  3.48  4.43  21.17  0.88  3.46  11.34  32.64  1.52  
   N  8  8  8  8  8  8  8  8  8  
 3 Mean   11.25  11 .25  0.00  72.74  6.7  10.38  6.84  97.04  9.54  
   Std  3.15  3.15  0.00  18.31  0.68  2.92  10.54  28.41  1.63  
   N  8  8  8  8  8  8  8  8  8  
 4  Mean  9.5  9.5  0.00  63.95  6.96  8.25  9.61  70.99  9.00  
   Std  4.31  4.31  0.00  32.63  2.15  3.24  12.57  22.22  1.5  
   N  8  8  8  8  8  8  8  8  8  

Table 4: Sex ratio of pups - Group mean data

             at birth           On day4 post partum 
 Group    M  F  Total  % males  M  F  Total  % males
 1  Mean  6.67  5.22  11.89  57.4  5.89  4.44  10.33  58 .79
   Std  1.00  2.11  1.83  12.66  1.90  2.30  3.24  13.14
   N  9  9  9  9  9  9  9  9
 2  Mean  4.63  4.50  9.13  52.08  4.50  4.13  8.63  53.81
   Std  1.60  2.51  3.48  10.93  1.60  2.42  3.46  11.53
   N  8  8  8  8  8  8  8  8
 3  Mean  5.63  5.63  11.25  51.30  5.13  5.25  10.38  50.93
   Std  2.72  2.72  3.15  21.20  2.47  2.60  2.92  20.61
   N  8  8  8  8  8  8  8  8
 4  Mean  4.50  5.00  9.50  46.05  4.00  4.25  8.25  46.15
   Std  2.78  2.56  4.31  14.97  2.51  1.67  3.24  16.06
   N  8  8  8  8  8  8  8  8

Applicant's summary and conclusion