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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013.11.18 - 2014.06.20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Dicumyl Peroxide; Chemical name: bis(α, α-dimethylbenzyl) peroxide
- Physical state: Crystalline, white
- Analytical purity: technically pure
- Composition of test material, percentage of components: bis(α, α-dimethylbenzyl) peroxide , technically pure
- Lot/batch No.: 113040311
- Expiration date of the lot/batch: August 20, 2014
- Storage condition of test material: Store in original package at room temperature (max.: 30 °C)
- Assay (peroxide content): 99.00%
- Active oxygen content: 5.86%

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd. Brl.Han: WIST
- Source: TOXI-COOP ZRT. 1103 Budapest, Cserkesz u. 90.
- Age at study initiation: Females 6-9 weeks; males 9-10 weeks
- Weight at study initiation: Females 100-160 g; males 250-310 g
- Fasting period before study:
- Housing: 2-3 males or females per cage, Type II polypropylene/polycarbonate with stainless steel covers equipped by self-feeding baskets, certified laboratory wood bedding changed twice a week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 64 days for females, 82 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22 °C
- Humidity (%): 30-59 %
- Air changes (per hr): 10-15 air exchanges/hour by central air-conditioning system.
- Photoperiod (hrs dark / hrs light): Artificial light, from 6 a.m. to 6 p.m.

IN-LIFE DATES: until 3 weeks after mating

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Dicumyl Peroxide was proved to be stable in sunflower oil in formulations at room temperature for up to 24 hours and at 5 ± 3 ºC for up to five days
- Amount of vehicle (if gavage): 2 ml/kg bw
- Lot/batch no. (if required): 1305-4630
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preparation of the test item formulation was made in the vehicle from daily up to every five days using magnetic stirrer in the formulation laboratory of the test facility. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male/1-3 females
- Length of cohabitation: 2-3 hours until the number of sperm positive females/group were achieved
- Proof of pregnancy: vaginal plug and/or sperm in the vaginal smear referred to as day 0 / day 1 of pregnancy
Duration of treatment / exposure:
Days 5-19 of gestation
Frequency of treatment:
Daily
Duration of test:
Necropsy on gestation day 20
No. of animals per sex per dose:
24 sperm positive females/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected with agreement of the sponsor based on the results of a dose range finding study by oral administration and literature data presented by the sponsor
- Rationale for animal assignment (if not random): The sperm positive females were allocated if possible to each experimental group on each mating day in such a way that the group averages of the body weight were as similar as possible on the first day of gestation. If possible, females paired with the same male were allocated to different groups on the same mating day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day

BODY WEIGHT: Yes (females only)
- Time schedule for examinations: once during pre-mating period, gestation days 0, 3, 5, 8, 11, 14, 17 and 20, corrected body weight was calculated for the 20th day of pregnancy

FOOD CONSUMPTION: Yes
- The food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: uterus with cervix and the left ovary, viscera
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: No
- Number of late resorptions: No
- Other: live fetuses, early and late embryonic death, foetal death
Fetal examinations:
- External examinations: Yes: all live foetuses per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
- viability, weight, gender, half subject to visceral examination
Statistics:
- Statistical analysis was performed with SPSS PC+ software.
- The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way ANOVA was carried out. If the obtained result was positive, Duncan's multiple range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way ANOVA was used. If there is a positive result, the intergroup comparisons are performed using the Mann-Whitney U-test. The chi²-test was performed where appropriate and feasible.
Indices:
Maternal: Pre-implantation loss, post-implantation loss
Fetus: Sex distribution, fetal body weight external abnormalities/litter, visceral abnormalities/litter, skeletal abnormalities/litter
Historical control data:
Historical control data was provided to allow comparison with current controls. Historical control data was taken from "Background pregnancy and fetal data from developmental toxicity studies in teh Hsd. Brl. Han: WIST Rat" from January 2014.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
450 mg/kg bw/day caused death, piloerection, reduced activity, coldness, paleness, vaginal bleeding and hypotonicity, enlarged adrenals and spleen and blood in the uterus, markedly reduced food consumption, lower body weight, markedly reduced body weight gain and weight loss as well as markedly reduced corrected body weight and body weight gain in the maternal animals.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
450 mg/kg bw/day resulted in increased postimplantation loss (and lower number of viable fetuses), a decreased foetal weight, an increased percentage of foetuses with body weight retardation, malrotated fore-and hindlimbs as well as skeletal malformations of the pectoral girdle and extremities, increase of skeletal variations and placentas with dark brownish discoloration or fibrinoid degeneration possibly due to the marked maternal toxicity.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
external malformations
skeletal malformations
other: placentas with dark brownish discoloration or fibrinoid degeneration possibly due to the marked maternal toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Pregnancy Data of Females, Mortality

Dose groups Control 50 mg/kg bw/day 150 mg/kg bw/day 450 mg/kg bw/day
Number of sperm positive females 24 24 24 24
Number of females with no implantation but corpora lutea 0 0 0 0
Number of females with no implantation and no corpora lutea 1 4 3 6
Number and percent of pregnant females (females with implantation) 23 20 21 18
Number of evaluated dams 23 20 21 18
Number of pregnant females died (due to toxicity) 0 0 0 1
Number of dams with total intrauterine death 0 0 0 0
Number of evaluated litters 23 20 21 17
Number and percent of evaluated litters with malformed fetuses 4 0 3 10

SUMMARY OF CLINICAL SIGNS AND NECROPSY FINDINGS OF DAMS (sum, %)          
DESCRIPTIONDOSES: No.
of animals:
control
23
50
mg/kg bw/day
20
150 mg/kg bw/day
21
450 mg/kg bw/day
18
MORTALITY          
- died due to an unclear reason N 0 0 0 1
  % 0 0 0 6
CLINICAL SYMPTOMS          
- none N 22 20 17 7
  % 96 100 81 39
- alopecia N 1 0 0 3
  % 4 0 0 17
- salivation N 0 0 4 8
  % 0 0 19 44
- piloerection N 0 0 0 4
  % 0 0 0 22
- reduced activity N 0 0 0 2
  % 0 0 0 11
- red coloration around red eye N 0 0 0 1
  % 0 0 0 6
- pale N 0 0 0 2
  % 0 0 0 11
- vaginal bleeding N 0 0 0 2
  % 0 0 0 11
- hypotonicity N 0 0 0 2
  % 0 0 0 11
- cold N 0 0 0 2
  % 0 0 0 11
NECROPSY FINDINGS          
- no macroscopic alterations N 23 20 21 11
  % 100 100 100 61
- enlarged adrenals N 0 0 0 5
  % 0 0 0 28
- enlarged spleen N 0 0 0 2
  % 0 0 0 11
- uterus filled up with blood N 0 0 0 1
  % 0 0 0 6
- bloody vaginal orifice N 0 0 0 1
  % 0 0 0 6
- blood in uterus N 0 0 0 3
  % 0 0 0 17
- pale liver N 0 0 0 1
  % 0 0 0 6
- pale kidneys N 0 0 0 1
  % 0 0 0 6
- stomach distended, N 0 0 0 1
filled up with darker content % 0 0 0 6

Body weight (g) of Dams (mean, SD)
TIME Gestational days DOSE GROUPS (mg/kg bw/day)   
  Control               50 150 450  
0          
MEAN 236 236.8 233.1 234.9  
SD 20.67 14.86 10.65 10.96  
N 23 20 21 18  NS
5          
MEAN 248.6 251 248.9 251.2  
SD 20.56 15.05 12.48 13.86  
N 23 20 21 18 NS
8          
MEAN 254.4 253.9 246.7 245.2  
SD 21.21 15.2 12.65 14.98  
N 23 20 21 18  
11          
MEAN 267.3 265.3 254.8 246.3  
SD 21.51 16.3 13.08 15.2  
N 23 20 21 * 18 **    DN
14          
MEAN 278.8 275.8 265.5 255.5  
SD 21.89 15.53 11.47 17.66  
N 23 20 21 * 18 **    U
17          
MEAN 301.1 297.8 286.4 274.2  
SD 22.36 16.74 12.06 18.76  
N 23 20 21 * 18 **    DN
20          
MEAN 338.7 335.8 321.2 283.6  
SD 27.59 20.72 14.54 24.5  
N 23 20 21 ** 17 **    U

SUMMARY OF BODY WEIGHT GAIN OF DAMS
Body weight gain (g) (mean, SD)
TIME Gestational days DOSE GROUPS (mg/kg bw/day)
    Control 50 150 450
0-5 MEAN 12.6 14.2 15.8 16.2
  SD 3.95 4.46 4.43 5.24
  n 23 20 21 * 18 * DN
5-8 MEAN 5.8 2.9 -2.2 -5.9
  SD 3.42 3.48 3.80 5.61
  n 23 O * 21 ** 18 ** DN
8-11 MEAN 12.8 11.5 8.1 1.1
  SD 3.60 3.61 5.32 7.77
  n 23 20 21 ** 18 ** U
11-14 MEAN 11.6 10.5 10.7 9.2
  SD 4.02 3.69 4.30 8.51
  n 23 20 21 18 NS
14-17 MEAN 22.3 22.1 20.9 18.7
  SD 3.99 3.93 3.79 7.75
  n 23 20 21 18 NS
17-20 MEAN 37.7 38.0 34.8 11.5
  SD 8.40 7.59 6.86 16.49
  n 23 20 21 17 ** U
0-20 MEAN 102.7 99.0 88.0 49.5
  SD 14.70 13.11 12.77 19.96
  n 23 20 21 ** 17 ** DN

SUMMARY OF FOOD CONSUMPTION DATA OF DAMS
(mean, SD)
TIME Gestational days DOSE GROUPS (mg/kg bw/day) 
Control 50 150 450  
0-3 MEAN 17.2 17.3 17.7 18.1  
  SD 1.84 2.18 1.41 1.56  
  n 23 20 21 18 NS
3-5 MEAN 19.8 20.5 20.7 21.5  
  SD 1.19 2.27 1.15 2.33  
  n 23 20 21 ** 18 ** U
5-8 MEAN 19.4 17.2 14.0 10.9  
  SD 1.37 2.16 1.53 1.43  
  n 23 20 ** 21 ** 18 ** DN
8-11 MEAN 19.4 17.7 14.5 11.5  
  SD 1.11 2.49 2.47 2.24  
  n 23 20 ** 21 ** 18 ** U
11-14 MEAN 20.5 19.1 16.6 13.9  
  SD 1.41 1.52 1.37 2.38  
  n 23 20 ** 21 ** 18 ** U
14-17 MEAN 20.9 19.7 17.6 16.7  
  SD 1.29 2.57 1.58 2.08  
  n 23 O * 21 ** 18 ** U
17-20 MEAN 22.0 21.8 18.9 13.9  
  SD 1.77 2.33 1.58 2.67  
  n 23 20 21 ** 18 ** DN

INTRAUTERINE MORTALITY, VIABLE FETUSES, SEX DISTRIBUTION (mean, SD)
GROUPS (mg/kg bw/day):    Control 50 150 450  
NUMBER OF DAMS:   23 20 21 18  
Corpora Lutea Mean: 13.5 12.9 12.4 12.8  
  SD: 1.75 1.37 1.77 1.38 NS
Preimplantation Loss % Mean: 7.4 11.7 8.4 13.7  
  SD: 11.97 15.45 10.20 13.58 NS
Implantation Mean: 12.5 11.4 11.3 11.0  
  SD: 2.23 2.35 1.79 1.71 NS
Early Embryonic Death % Mean: 5.9 3.3 4.2 1.7  
  SD: 6.85 4.95 6.19 3.98 NS
Late Embryonic Death % Mean: 1.3 1.0 0.8 11.8  
  SD: 4.58 3.13 2.40 14.00 ** U
Dead Fetuses % Mean: 0.0 0.0 0.0 3.2  
  SD: 0.00 0.00 0.00 7.27 ** DN
Postimplantation Loss % Mean: 7.2 4.4 5.0 16.8  
  SD: 8.71 5.99 6.47 17.44 ** U
Total Intrauterine Mortality % Mean: 14.2 15.6 12.9 28.1  
  SD: 13.37 15.68 11.88 19 77 ** DN
Viable fetuses Mean: 11.6 10.9 10.7 9.0  
  SD: 2.35 2.38 1.79 2.57 ** DN
Male fetuses % Mean: 43.9 51.7 48.2 50.4  
  SD: 17.01 14.66 14.49 14.06 NS
Female fetuses % Mean: 56.1 48.3 51.8 49.6  
  SD: 17.01 14.66 14.49 14.06 NS

RESULTS OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS (percentile litter means and SD)
    DOSE GROUPS (mg/kg bw/day)
    Control 50 150 450  
EXTERNAL EXAMINATION            
Litters examined N 23 20 21 17  
Fetuses examined N 266 218 225 153  
Fetuses Mean 2.5 2.3 3.5 26.2 ** U
with abnormalities SD 5.46 4.12 5.59 24.34  
Variation Mean 2.5 2.3 3.5 21.5 ** U
  SD 5.46 4.12 5.59 24.62  
Malformation Mean 0.0 0.0 0.0 4.7 ** DN
  SD 0.00 0.00 0.00 9.03  
Retarded in body weight Mean 2.5 2.3 3.5 22.2 ** U
  SD 5.46 4.12 5.59 23.72  
VISCERAL EXAMINATION            
Litters examined N 23 20 21 17  
Fetuses examined N 133 109 111 77  
Fetuses Mean 1.3 2.0 1.0 2.0 NS
with abnormalities SD 4.47 8.94 4.36 5.78  
Variation Mean 0.0 2.0 0.0 2.0 NS
  SD 0.00 8.94 0.00 5.78  
Malformation Mean 1.3 0.0 1.0 0.0 NS
  SD 4.47 0.00 4.36 0.00  
SKELETAL EXAMINATION            
Litters examined N 23 20 21 17  
Fetuses examined N 133 109 114 76  
Fetuses Mean 19.4 15.0 22.7 61.4 ** DN
with abnormalities SD 21.32 24.29 31.81 30.69  
Variation Mean 17.8 15.0 19.9 39.8 ** DN
  SD 19.61 24.29 27.56 23.91  
Malformation Mean 1.6 0.0 2.9 21.6 ** U
  SD 5.31 0.00 9.56 28.75  

LITTER MEANS OF FETAL AND PLACENTAL WEIGHT
    DOSE GROUPS (mg/kg bw/day)
    Control 50 150 450
    M+F M F M+F M F M+F M F M+F M F
Fetal weight MEAN 3.3 3.4 3.2 3.3 3.3 3.2 3.3 3.4 3.2 2.9 3.0 2.9
(g) SD 0.18 0.16 0.20 0.19 0.24 0.19 0.19 0.18 0.23 0.29 0.29 0.32
  n 23 23 23 20 20 20 21 21 21 Y]** 17 ** 17 **
                      DN U DN
Placental weight MEAN 717.0 721.7 713.3 699.9 699.0 698.7 690.1 695.3 681.8 690.6 693.6 686.1
(g) SD 48.84 64.10 51.88 56.31 59.40 68.85 45.72 55.68 43.55 90.37 120.89 90.17
  n 23 23 23 20 20 20 21 21 21 17 17 17
Relative placental weight MEAN 219.7 213.1 224.5 214.4 211.2 218.0 212.3 206.5 217.4 236.5 233.7 238.6
(mg/g) SD 16.3 17.06 23.25 16.41 20.00 20.00 17.11 17.43 18.67 32.19 40.73 34.39
  n 23 23 23 20 20 20 21 21 21 17 17.0 17.0

Applicant's summary and conclusion

Conclusions:
Treatment with dicumyl peroxide resulted in maternal and developmental toxicity at a dose of 450 mg/kg bw/day when administered during days 5-19 of gestation to rats. The developmental effects are possibly secondary to maternal toxicity. This conclusion has been supported by an independent re-evaluation conducted by an external pathologist.
Executive summary:

In a developmental toxicity study according to OECD 414, dicumyl peroxide was administered to 24 pregnant female Hsd. Brl. Han: WISTAR rats per dose by oral gavage at dose levels of 0, 50, 150 and 450 mg/kg bw/day from day 5 through 19 of gestation. The highest administered dose elicited pronounced maternal toxicity, including death, piloerection, reduced activity, coldness, paleness, vaginal bleeding and hypotonicity, enlarged adrenals and spleen and blood in the uterus, markedly reduced food consumption, lower body weight, markedly reduced body weight gain and weight loss as well as markedly reduced corrected body weight and body weight gain.

Effects of the highest dose on embryos included increased post implantation loss (and lower number of viable foetuses), a decreased foetal weight, an increased percentage of foetuses with body weight retardation, malrotated fore- and hindlimbs as well as skeletal malformations of the pectoral girdle and extremities, increase of skeletal variations and placentas with dark brownish discoloration or fibrinoid degeneration possibly due to the marked maternal toxicity.

 

The maternal LOAEL is 450 mg/kg bw/day.  The maternal NOAEL is 150 mg/kg bw/day. 

The developmental LOAEL is 450 mg/kg bw/day. The developmental NOAEL is 150 mg/kg bw/day.

The developmental toxicity study in the rat is classified as acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats. Considering the high incidence of skeletal malformation in the high dose group and some ambiguous effects in the mid-dose group, the study results have been re-evaluated by an external pathologist. The result of the re-examination confirmed that the skeletal findings critical to the result of this study were essentially reliable. Thus, the previously set NOAEL for developmental toxicity at a dose of 150 mg/kg bw/day in the original report can be considered acceptable.