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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:
2013-11-18 to 2014-06-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(α,α-dimethylbenzyl) peroxide
EC Number:
201-279-3
EC Name:
Bis(α,α-dimethylbenzyl) peroxide
Cas Number:
80-43-3
Molecular formula:
C18H22O2
IUPAC Name:
1,1'-(dioxydipropane-2,2-diyl)dibenzene
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%
Specific details on test material used for the study:
- 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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
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.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system 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: not specified
- 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 : ad libitum ,ssniff® SM R/M-Z+H complete diet (ssniff Spezialdiäten GmbH, D-59494 Soest Germany)
- Water : tap water, ad libitum
- Acclimation period: 64 days for females, 82 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 30-59
- Air changes (per hr): 10-15 air exchanges/hour by central air-conditioning system.
- Photoperiod (hrs dark / hrs light): 12/12

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
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
concentration 25 mg/mL
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
concentration 75 mg/mL
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
concentration 225 mg/mL
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
Blood sampling:
not reported
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

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection, reduced activity, paleness, vaginal bleeding, hypotonicity and coldness were noted for females in the 450 mg/kg bw/day dose group which was attributed to an effect of the test item. Salivation was recorded for dams in the 450 and in the 150 mg/kg bw/day group which was judged to be in association with the treatment however as non adverse. No clinical observations were noted for the dams in the 50 mg/kg bw/day dose group
Mortality:
mortality observed, treatment-related
Description (incidence):
One dam died before scheduled necropsy in the 450 mg/kg bw/day dose group which was considered to be due to an effect of the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A lower mean body weight and corrected body weight of the pregnant females as well as a weight loss and a moderately to markedly lower body weight gain and corrected body weight gain of the pregnant females was observed in the 150 and 450 mg/kg bw/day groups which was attributed to the treatment, however these changes were judged not to reach an adverse degree in case of the animals in the 150 mg/kg bw/day dose group considering the reversibility of the body weight gain reduction and the slight degree of the body weight and corrected body weight A reduced body weight gain of the females was calculated in the
50 mg/kg bw/day dose group just for the first three days of the treatment. This was judged to be likely incidental concerning the small difference and that thereafter the body weight development of the females was at the control level and there was no difference relative to the control in the body weight and corrected body weight of the females during the whole in-life phase.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a moderate to marked dose related decrease in the food consumption of pregnant females to see in the 150 and 450 mg/kg bw/day groups respectively in the whole treatment period which was ascribed to the treatment. The food consumption reduction in the 150 mg/kg bw/day dose group was judged to be below an adverse level and biologically non relevant.
A slight reduction in the food consumption was indicated in the 50 mg/kg bw/day dose group during the treatment period, however this statistically significant but toxicologically irrelevant change was considered to be within the biological variation and not proven to be due to the test item.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic findings at necropsy such as enlarged spleen and adrenals as well as bloody orifice and bloody uterine content in the 450 mg/kg bw/day dose group were found to be a result of the treatment with Dicumyl Peroxide. There were no macroscopic findings observed in the dams in the 50 and 150 mg/kg bw/day dose groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Postimplantation loss (due to late embryonic and fetal death) increased, the number of viable fetuses decreased significantly in the 450 mg/kg bw/day group which was considered to be an adverse effect due to the treatment with the test item.
Preimplantation loss was statistically significantly higher in the 450 mg/kg bw/day group than in the control, however without a clear dose response.
Total litter losses by resorption:
not specified
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
The number of viable fetuses decreased significantly in the 450 mg/kg bw/day group which was attributed to an effect of the test item.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Other effects:
not specified
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
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
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)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight of the male and female fetuses was statistically significantly lower in the 450 mg/kg bw/day dose group if compared to the control and this was attributed to an effect to the test item. The mean weight of the fetuses in the control, 50 and 150 mg/kg bw/day groups was identical.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Postimplantation loss (due to late embryonic and fetal death) increased, the number of viable fetuses decreased significantly in the 450 mg/kg bw/day group which was attributed to an effect of the test item
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Anogenital distance of all rodent fetuses:
not specified
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
Body weight retardation (below 2.84 g for males and 2.54 g for females) was categorized as an external variation. The incidence of body weight retarded fetuses increased markedly in the 450 mg/kg bw/day dose group which was attributed to an effect due to the treatment of the dams. There were no significant differences in the incidence of body weight retardation of fetuses in the 50 and 150 mg/kg dose group.
External malformations malrotated fore and/or hindlimbs in 6 fetuses (mainly associated with skeletal findings) were found in the 450 mg/kg bw/day group which was judged to be in association with the treatment of the dams with the test item.
No fetal malformations found at external examination in the control, 50 and 150 mg/kg bw/day dose groups.
Placental findings as dark brownish discoloration of the margin of the placentas were found in 44 and fibrinoid degeneration was recorded in 11 cases in the 450 mg/kg bw/day group which was attributed to an effect of the test item. There were no placental findings observed in the control, 50 and 150 mg/kg bw/day dose group.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There was a significant increase in the incidence of fetuses with skeletal malformations and variations in the 450 mg/kg bw/day group.
Malformations:
Malformations such as short and/or bent scapula, clavicula, humerus, radius, ulna, femur, tibia and fibula occurred only in the 450 mg/kg bw/day group.
Short and/or bent scapula, humerus and radius occurred with a higher incidence than recorded in the historical control database. The incidence of short and/or bent femur, tibia and fibula was within the historical control range. Malformations of the pectoral girdle and extremities in the 450 mg/kg bw/day dose groups were considered to be in association with the treatment of the dams with the test item.
Variations:
Significanty increased the occurrence of incomplete ossification of skull bones (markedly incomplete ossification of one or more bone), the incompletely ossified sternum, metacarpal/metatarsal and wavy and markedly wavy ribs in the 450 mg/kg bw/day dose group which was considered to be in association with the treatment, possibly due to maternal toxicity.
In a re-evaluation of the findings, it was suggested that these findings might not only be reversible but also of short duration and not harmful to the animal in the longer term. With this in mind, it can be argued that the critical girdle and limb findings in this study could all be classified as variations and, in the absence of other major foetal abnormalities at this dose level, it could be suggested that the NOAEL for developmental toxicity could be raised to 450 mg/kg.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The visceral variation hydroureter (bilateral) was found in the 50 and mg/kg bw/day and 450 mg/kg bw/day groups with a low incidence (2 in both groups) without a dose response.
This variation was considered to be without a relationship to the treatment.
Other effects:
not specified
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
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Key result
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

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: clavicle
skeletal: scapule
other: humerus, radius and ulna
Description (incidence and severity):
Skeletal malformations such as short and/or bent scapula, clavicula, humerus, radius and ulna occurred in the 450 mg/kg bw/day group with a higher incidence than recorded in the historical control database. This change was considered to be due to an effect of the test item.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
450 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Relevant for humans:
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.