Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The registrant provided data from a 90-d study (OECD 408) with detailed investigations of reproductive organs, oestrous cycle and sperm parameters, as well as for a developmental toxicity study in the rat according to OECD guideline 414. The results of the two studies indicate no need for further information. The risk assessment is driven by general toxicity with a NOAEL of 80 mg/kg/d and physico-chemical properties (organic peroxide Type F). This approach takes into account animal welfare considerations to the maximum possible extent.


Short description of key information:
Developmental toxicity study in rat (OECD 414); 90-d study (OECD 408) with detailed investigations of reproductive organs, oestrous cycle and sperm parameters.

Effects on developmental toxicity

Description of key information

GLP OECD 414 study in rats: Foetal malformations were found at the highest dose level (450 mg/kg bw/d), which is also the LOAEL for maternal toxicity, with marked toxic effects. It was concluded that the observed foetal malformations are secondary effects. The assessment has been re-evaluated by an independent 3rd party pathologist.

GLP OECD 414 study in rabbit: No indication of developmental or foetal toxicity observed up to highest dose tested. The NOAEL for maternal toxicity is considered to be 50 mg/kg bw/day. At HD, dead or moribund sacrificed females were considered due to the severe local disturbance of the functioning of the gastro-intestinal tract. Based on number of terminally sacrificed females, NOAEL for foetal toxicity is considered to be 150 mg/kg bw/day (MD).

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
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
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.
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.
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.
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
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.
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
Abnormalities:
not specified
Developmental effects observed:
not specified

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
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.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-01-28 to 2020-02-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Name: Bis(a,a-dimethylbenzyl)peroxide
- CAS No.: 80-43-3
- Batch No.: 11712271
- Physical state: solid
- Colour: white
- Purity: 99.9%
- Storage conditions: room temperature
- Expiry date: October 2019

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was dissolved in sunflower oil. The vehicle had been selected based on the test item’s characteristics and testing guideline. The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item, further vortexing it for 45-60 minutes.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 19-25 weeks old
- Weight at study initiation: males: 3108 – 4143 g; females: 2695 - 4784 g
- Fasting period before study: none
- Housing: Semi barrier in an air-conditioned room, housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm²
- Diet (e.g. ad libitum): Free access to autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Water (e.g. ad libitum): Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least 5 days acclimatisation period under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature: 18 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
The test item formulation was prepared at least once every 10 days. The prepared formulation was stored protected from light and at room temperature. The vehicle was also used as control item. The test item formulation or vehicle was administered at a single dose to the animals by oral gavage. The application volume for all groups was 1.5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preliminary stability analysis was performed on the samples from high dose and low dose groups and the investigation was made at the following time points (temperature) after dose formulation: 0 h, 6 h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day (-15 to -35 °C). Preliminary homogeneity investigation was performed on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups. As the test item was shown to be homogenous according to Eurofins Study No. 182674 (after 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples were taken for substance concentration in the first, third, fifth and last weeks of the study for all doses including the vehicle control (16 samples in total). Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 182675) and until then stored under appropriate conditions based on available stability data. The B-samples are retained at below -15 °C at BSL Munich (test facility) and discarded after completion of the final study report.
Details on mating procedure:
Females were paired in batches in order to regularise the number of animals for terminal sacrifice on a particular day. Animals were paired in 1:1 ratio (male to female). At the time of pairing, a female was taken to a male’s cage. Mating was confirmed by visual inspection and based on the falling of male on its back or side and emits of typical cry. In case mating was unsuccessful the female was taken back to its original cage and mating of the female with another male or at another time was considered. All females mated on a single day were distributed in all groups based on their body weight and it was ensured that each day mean group body weight was comparable. The day on which a confirmed mating was observed visually is called GD ‘0‘ of presumed pregnancy.
Duration of treatment / exposure:
between gestation day 6 and 27
Frequency of treatment:
once daily during GD 6 and GD 27
Duration of test:
Animals were sacrificed on the respective GD 28.
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control group
Dose / conc.:
20 mg/kg bw/day
Remarks:
Low Intermediate Dose (LID) group
Dose / conc.:
50 mg/kg bw/day
Remarks:
Low Dose (LD) group
Dose / conc.:
150 mg/kg bw/day
Remarks:
Medium Dose (MD) group
Dose / conc.:
250 mg/kg bw/day
Remarks:
High Dose (HD) group. (From treatment day 22, the dose level was reduced from 325 mg/kg bw to 250 mg/kg bw).
Dose / conc.:
325 mg/kg bw/day
Remarks:
High Dose (HD) group
No. of animals per sex per dose:
For groups: Control, LID, LD and MD: 20 animals/dose group
For group: HD: 16 animals
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The study design was based on the DRF study (Eurofins study no. 182672, non GLP). In the dose range finding study (Eurofins study no. 182672, non GLP) pregnant New Zealand White rabbits were treated with the test item at doses of 100, 200 and 300 mg/kg bw/day from GD 6 to 27.
During the period of administration, the animals were observed precisely each day for signs of toxicity and mortality. Animals that died during the study were examined macroscopically. All surviving female animals were sacrificed on GD 28. Following the gross necropsy, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late) as well as live and dead foetuses. Foetuses were identified using numbered plates attached to strings tied around the neck or the abdomen, sexed and weighed. All foetuses were observed for external abnormalities.
Body weight and food consumption were measured on GD 0, 3, 6, 9, 12, 15, 18, 21, 24 and 27.
The uteri of the non-pregnant females were processed with 10 % ammonium sulphide solution and checked for the early embryonic deaths.

Results:
No test item-related mortality was observed in this study. However, one female from control (no. 3) on GD 19 and 1 female from HD (no. 22) group on GD 10 were found dead in this study. Macroscopic evaluation of the HD female at necropsy revealed discoloured, dark, fluid-filled lung indicative of gavaging error, which was not considered to be related to the treatment with the test item.
No test item-related clinical signs were observed in females of any treated groups. A few predominant spontaneous clinical signs like hairless area on various body parts (2 out of 6 animals in control as well as in MD groups), crust on right hindpaw or forepaw (1 out of 6 in control as well as in LD groups) and reduced spontaneous activity (1 out of 6 in MD group) were observed for a few days. Although no toxicologically relevant clinical signs were observed in the study, 2 females (1 each from MD and HD groups) showed signs of abortion and 1 female from MD group had premature delivery prior to the scheduled sacrifice.
The mean body weight and body weight gain were comparable between the control and all treatment groups before initiation of the treatment up to GD 6. However, mean body weight and mean body weight gain were slightly lower in the MD and HD groups compared to the control group starting from GD 9 onwards throughout the study period. However, this decrease in body weight was not consistent as study progressed and was within 10 % variation compared to the control group.
In correlation to the body weight and body weight change, food consumption was noted to be lower during GD 6-12, 15-21 and 24-27 for the MD group and GD 6-18 for the HD group compared to the control group. The overall mean food consumption throughout the study period (GD 0-28) was moderately lower in MD and HD groups when compared to the control group. However, there was no dose-response relationship observed and the food consumption decrease was also transient such that no decrease was observed in the HD group towards the end of the study (GD 18-27).
Successful mating resulted in 6/6 pregnancies in the control and HD group, 5/6 pregnancies in LD and 4/6 in MD groups.
Prenatal parameters such as group mean number of corpora lutea, implantation sites, early or late resporptions, number of female foetuses, pre- and post-implantation losses remained unaffected in all dose groups when compared to the control group. No dead foetuses were observed in any of the groups.
A moderately lower maternal terminal body weight, gravid uterus weight, adjusted maternal body weight (i.e. terminal body weight minus gravid uterus weight), number of male foetuses, sex ratio and number of live foetuses were observed in the MD and HD groups when compared to the controls.
No relevant treatment-related changes of toxicological relevance were observed for parameters like mean foetus weight, number of female foetuses and female litter weight in any of the treatment groups when compared with the controls. However, total number of foetuses, total litter weight, number of males and male litter weight were marginally lower in the all treatment groups, especially in the HD group, when compared to the control group. However, as the difference of these parameters was marginal, it cannot be clearly determined in this study whether these changes are related to treatment with the test item.
Foetal external examination on the day of terminal sacrifice revealed no external findings in foetuses of any of the treated or control groups.
No gross pathological changes were observed during the macroscopic observation of terminally sacrificed does of all dose groups on GD 28. However, one HD doe found dead was observed with discoloured dark and fluid filled lungs attributed to gavaging error.

Conclusion:
No test item-related toxicological effects in terms of clinical signs, mortality or gross pathological findings in does as well as foetal external findings were observed in any of the treatment groups when compared with the controls. Two does (1 each from the MD and HD groups) showed signs of abortion and 1 dose from the MD group had premature delivery prior to the scheduled sacrifice. Nevertheless, no toxicologically relevant clinical signs were observed in the study. There were transient effects on body weight and food consumption as well as slight effects on several reproductive and foetal parameters, such as maternal terminal body weight, gravid uterus weight, adjusted maternal body weight, number of male foetuses, sex ratio, number of live foetuses, total litter weight and male litter weight, observed in the MD and HD groups when compared with the control group.
No findings of toxicological relevance were observed for any maternal or foetal parameters of LD group when compared with the controls. However, in this dose range finding (DRF) study, no foetal visceral, skeletal and craniofacial evaluations were included. These evaluations will be part of the main study and therefore in order to establish an overall NOAEL for the main study (in case there are foetal pathology findings at 100 mg/kg bw) a LD below 100 mg/kg body weight/day is proposed. Based on the data generated from this DRF study, dose levels of 50, 150 and 325 mg/kg body weight/day were proposed for the main prenatal developmental toxicity study.

Justification for inclusion of Low Intermediate Dose (LID) instead of additional high dose group:
In the main study the following doses where administered, 20, 50, 150 and 250/325 mg/kg bw/d. In the main study two mortalities (female 85 on GD 12 and 96 on GD 6) and 13 early sacrifices (female 76 on GD 6, 77, 79 and 98 on GD 15, 78 on GD 23, 86 on GD 14, 87, 90 and 99 on GD 21, 91 and 95 on GD 14 and 94 on GD 11) due to moribund conditions from HD group were considered to be due to the severe local disturbance of the functioning of the gastro-intestinal tract the high dose 325 mg/kg bw/d.
Due to the severe effects of treatment with 325 mg/kg bw/d, the high dose level was reduced to 250 mg/kg bw/d on GD 22.
The OECD TG 414 states that the "highest dose should be chosen with the aim to induce some developmental and/or maternal toxicity (clinical signs or a decrease in body weight) but not death or severe suffering." Therefore, regarding animal welfare reasons in light of the severe effects which were observed even at the reduced high dose level, it is not justified for the contract research organization to include another dose level between 250 mg/kg bw/d and the already existing level of 150 mg/kg bw/d without causing additional severe suffering of the respective animals.
Considering the recommendations of the OECD TG 414, a two- to four-fold lower interval (2.5 x) was chosen for the LID (20 mg/kg bw/d) of the main study in order to increase the likelihood of generating dose-related data that includes a dose at the NOAEL using three dose level in the main study.
At 150 mg/kg bw/d, the mid dose level reduced body weight and food consumption were observed. The aim to induce some developmental and/or maternal toxicity was met, for details see the results section.
The overall aim of dose selection is to establish an appropriate dose-related response in order to derive a no observed adverse effect level (NOAEL). There should be clear signs of toxicity to ensure the potential hazard of the test item is assessed. Therefore, as described in the respective results sections of the dossier, it can be concluded that this dose level evoked adverse effects in animals sufficient to fulfill the requirements of the OECD TG 414.



Maternal examinations:
- Cage-Side Observations: Yes
General clinical observations were made at least once a day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

- Detailed Clinical Observations: Yes
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

- Body Weight: Yes
All animals were weighed once before initiation of pairing to ensure that the body weights are within ± 20% variation. The mated females were weighed during gestations days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28. Males were not weighed in this study except once before initiation of pairing.

- Food Consumption: Yes
Food consumption of pregnant females was measured on gestations days 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28. Food consumption was not measured for males during the entire study or for both male and females during the mating period.

- Post Mortem Examination: Yes
On GD 28, presumed pregnant females (does) were subjected to a caesarean section after sacrificing the animals using an overdose of sodium pentobarbital. Females showing signs of abortion or premature delivery prior to scheduled termination were sacrificed and subjected to a thorough macroscopic examination. At the time of termination or death during the study, the doe was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings were preserved in 4% neutral-buffered formaldehyde. Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the does was confirmed. Uteri that appeared non-gravid were further examined by staining with 10% ammonium sulphide solution to confirm the non-pregnant status. Males were sacrificed without any observations at any time after the completion of the mating or were used for other studies.
Ovaries and uterine content:
Each gravid uterus with the cervix was weighed. However, the gravid uterus obtained from dead animals was not weighed. The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or foetal deaths as well as the number of viable foetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of foetuses in each uterine horn was also recorded. Males were sacrificed without any observations at any time after the completion of the mating or were used for other studies.
Fetal examinations:
- External examinations: Yes, all per litter
Lip and palate were examined for cleft lip and palate by gently opening the mouth with forceps. The head, eyes, ears, jaw and snout was examined for the shape and size. The trunk was examined for any external abnormalities. Limbs were examined for shape, size, position and digits for number and depth of digital furrows. The tail was examined for presence, size, shape and position.

- Soft tissue examinations: Yes, all per litter
Foetuses for the visceral examination were examined with the ventral side up under a stereo microscope. The abdominal and thoracic cavities of all foetuses were dissected and examined for visceral anomalies. The intestine, stomach, spleen and pancreas were examined for size and position. The liver was examined for size, shape, colour and number of lobes. The kidneys were further observed for the presence of clear fluid-filled cysts, cortical cysts, pitting or granular appearance and then sectioned with a sharp scalpel blade to examine the pelvis for distention or the presence of calculi or white granular material. The left kidney was sectioned with one longitudinal slice just off center and the right kidney was sectioned with one transverse slice directly through the papilla. The capsule, cortex, medulla, renal papilla, and renal pelvis were checked for the presence and the pelvis for distension with fluid. The reproductive organs were exposed by raising the intestine and the attached viscera from the dorsal wall and examined for any developmental defect. The rib cage was cut from the side of the sternebrae and xyphisternum (6th sternebra) to examine the thoracic organs. The lung was observed for size, colour and number of lobes. The thymus gland was checked for size and position. The trachea and oesophagus were exposed by removing the thymus gland and examined for fusion or tracheaoesophageal fistula. The position, size, colour and shape of the heart were observed. The pericardial sac was opened and the heart was fully exposed and examined for the presence or absence of major blood vessels like aortic arch, pulmonary artery and ductus arteriosus. For an examination of the internal anatomy of the heart, the heart was then repositioned and two cuts through the right ventricle were made using micro-dissecting scissors. The first cut was taken starting from the right of the ventral midline surface at the apex to the base of the pulmonary artery and the second cut was made through the midline surface at the apex extending to the left ventricle into the ascending aorta. Incisions were opened with fine forceps for the examination of interventricular and auriculo-ventricular septum.

- Skeletal examinations: Yes, all per litter
For skeletal examination, all foetuses were skinned after evisceration. Foetuses from entire litter were transferred into plastic bottles containing 94-95 % ethanol. These foetuses were processed using the Alizarin red staining technique. After fixation in 94-95 % ethanol, the foetuses were macerated with a 0.6 % aqueous potassium hydroxide solution for 10-13 days depending on maceration process and transferred to an Alizarin red solution (0.015 %) for 1 day. After that the foetuses were transferred to 25 %, 50 % and 75 % glycerine for one day each. Before examination the skeletons were fixed more than week in 86-89 % glycerin (clearing solution). The stained foetuses were examined under the stereomicroscope, the skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and tympanic ring (annulus). Similarly, the vertebral centres, ribs and sterna centres were also examined for size, shape and counted for the number of ossification centers. The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centers and arches. Pelvic girdles, fore limbs and hind limbs were examined for the development of the bones. Any deviation from the normal development was recorded for each foetus.

- Head examinations: Yes, half per litter
The head of the foetus was subjected to 5-7 sections in order to observe the internal structures of the head including the symmetry of the external nares, nasal conchae, nasal septum, palate, the development of the cerebellum and brain stem. Transverse
sections of the cephalic region were observed under the stereomicroscope and any anomalies were recorded.
Statistics:
All results were reported in tabular form (summarised in mean or summary tables and/or listed in individual data tables). Toxicology and pathology data were captured either on paper according to appropriate SOPs or using the validated computerised system Ascentos® System (version 1.3.4, Pathology Data Systems Ltd).A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Fisher’s exact test. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p< 0.05 is considered as statistically significant).
Indices:
The number of live births and post implantation loss, the number of pups with grossly visible abnormalities, the number of implantations, corpora lutea, live and dead foetuses and resorptions, litter size and litter weights were examined.
Historical control data:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In HD group females, in addition to reduced food intake, test item related reduced spontaneous activity was observed. All clinical signs observed in terminally sacrificed females up to MD group were incidental or non-adverse in nature.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Moribund sacrifice of one each female from LID and LD was attributed to gavaging error. Two mortalities and 13 morbidities from HD group were considered to be due to the severe local disturbance of the functioning of the gastro-intestinal tract at this dose level. Complete HD group (except terminally sacrificed female No. 89) was sacrificed before scheduled terminal sacrifice. From treatment day 22, the dose level of HD group was reduced from 325 mg/kg bw to 250 mg/kg bw. See table 5 for a summary.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The statistical analysis of group mean body weight data revealed no statistically significant effect in treatment groups except for the MD group. Statistically significantly lower group mean body weights were observed at the end of the treatment period on GD 27 (mean: 3,850.3 g (-7.5 %)) in MD group when compared to the control. A statistically significantly lower group mean body weight gain was observed during gestation days 12-15 (-48.2 g), 24-27 (-73.7 g), 0-27 (-107.3 g) in LD. In the light of no effect on group mean body weight, lack of consistency and individual data variation with only few animals with negative values contributed in lower group means, this effect on group mean body weight change in LD group was not considered to be test item related. However, the effect of significantly lower group mean body weight gain during gestation days 6-9 (-208.5 g), 9-12 (-85.6 g), 24-27 (-51.1 g) and 0-27 (-278.8 g) in the MD group can be considered test item related especially in correlation with reduced food consumption additionally observed in MD group which could be attributed to the severe local disturbance of the functioning of the gastrointestinal tract at this dose level. Therefore, the effects on body weight and weight changes of the MD group should be regarded as adverse. Food consumption and compound intake (if feeding study): In correlation to the body weight and body weight change, food consumption was noted to be statistically significantly lower during GD 6-9 (-71.3 %), 9-12 (-75.9 %) and 24-27 (-41.6 %) in the MD group compared to the control group. In addition, an increasing number of animals (16) needed to be provided with mash through syringes. This effect on food consumption in MD group was considered to be due to the severe local disturbance of the functioning of the gastrointestinal tract induced by this dose level and should therefore be regarded as adverse. No statistically or toxicologically relevant effect on food consumption was observed in other treatment groups. when compared with the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In correlation to the body weight and body weight change, food consumption was noted to be statistically significantly lower during GD 6-9, 9-12 and 24-27 in the MD group compared to the control group. This effect on food consumption in MD group was considered to be due to the severe local disturbance of the functioning of the gastro-intestinal tract at this dose level. No statistically or toxicologically relevant effect on food consumption was observed in other treatment groups. when compared with the controls.
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:
effects observed, treatment-related
Description (incidence and severity):
No test item related gross pathological changes of toxicological significance were observed during the macroscopic examination of the terminally sacrificed females of the control, LD, MD and HD groups. In LD group and LID moribund females (Nos. 41 and 17, respectively), gross evaluation of these females at necropsy revealed discoloured dark and fluid-filled lung indicative of gavaging error which was not considered to be related to the treatment with the test item. At macroscopic evaluation of the majority of dead or moribund sacrificed HD females at necropsy, major findings observed were fluid filled thoracic cavity, dark fluid filled and abnormal lung colour, gas filled colon, enlarged and abnormal coloured gallbladder which were considered were considered due to the severe local disturbance of the functioning of the gastro-intestinal tract.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Five females (3 from LD, 1 from LID and 1 from MD) showed signs of abortion prior to the scheduled sacrifice. Due to lack of dose dependency, this effect was not considered as test item related.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related effects of statistical or toxicological relevance were noted for percent pre- and post-implantation loss in treatment groups when compared to the controls.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related effects of statistical or toxicological relevance were noted.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related effects of statistical or toxicological relevance were noted for early or late resorptions in treatment groups when compared to the controls.
Dead fetuses:
no effects observed
Description (incidence and severity):
No test item-related effects of statistical or toxicological relevance were noted for number of live and dead foetuses, number of male and female foetuses in treatment groups when compared to the controls.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No test item-related effects of statistical or toxicological relevance were noted.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Successful mating resulted in 20/20 pregnancies in the control group 16/20 pregnancies in LD, 18/20 in LID, 17/20 in MD groups and 1/1 in HD group. Slightly lower pregnancy rates in the treatment groups were considered to be a biological variation and not related to the treatment with test item.
Other effects:
no effects observed
Details on maternal toxic effects:
PRENATAL DATA:
No test item-related effects of statistical or toxicological relevance were noted for any prenatal parameters including adjusted maternal weight (carcass weight), uterus weight, net weight change from GD 0, number of corpora lutea, implantation sites, early and late resorptions, number of live and dead foetuses, number of male and female foetuses, sex ratio and percent pre- and post-implantation loss in treatment groups when compared to the controls.

For detailed information on prenatal/litter data, please refer to Table 4 in section "Any other information on results incl. tables".
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
Remarks on result:
other:
Remarks:
The effects considered due to the severe local disturbance of the functioning of the gastro-intestinal tract at the HD.
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: gastro-intestinal tract
Description (incidence and severity):
The effects considered due to the severe local disturbance of the functioning of the gastro-intestinal tract at the HD.
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects of toxicological relevance observed for the mean foetus weight, male and female foetus weight on litter basis (group mean of individual litter mean) in any of the treatment groups including HD group when compared with the controls (for detailed results please refer to Table 3 in box "Any other informationon results incl. tables").
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No test item-related effects of statistical or toxicological relevance were noted for any prenatal parameters including number of live and dead foetuses when compared to the controls.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related effects of statistical or toxicological relevance were noted for any prenatal parameters including sex ratio when compared to the controls.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test item-related effects of statistical or toxicological relevance were noted.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Foetal external examination on the day of terminal sacrifice revealed no test item related external findings in foetuses of any of the treated groups.
Statistical analysis of data revealed no significant differences compared to the control group.
Low incidences of findings like gastroschisis (1 in control), eye bulge absent (1 in control), hindlimb limb malrotated (1 in control), head misshapen (1 in LID), tail short (1 in control and HD), hemimelia (1 in control) and forelimb malrotated (1 in control) were noted in isolated foetuses of the control group and/or the dose groups without dose dependency. As these findings were observed mostly in single foetuses, they were considered to be incidental in nature and unrelated to treatment with the test item.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examination of the foetuses after the Alizarin red staining technique for skeletal findings revealed a range of findings which were of a type or which occurred at an incidence generally comparable to or slightly lower or higher in the dose groups when compared to the control group. A statistically significant decrease in litter incidence for supernumerary ossification of sacral vertebral arch in all treatment groups compared to the control group was considered to be incidental as frequencies were even less in numbers compared to controls. Therefore, this finding is not to be considered as treatment-related and solely spontaneous in nature. A significantly higher litter incidence of unossified 5th sternebra was observed in LD, LID and MD group with statistical significance achieved only in LD group when compared to the control. Furthermore, there were marginally higher litter incidences of unossified talus observed in all treatment groups without achieving statistical significance (26.7% in LID, 50 % in LD, 33.3 % in MD when compared to 10 % in the controls). Generally delayed ossifications or incomplete ossifications of bones are considered to be variations. They are not regarded to persist postnatally and are not associated with long term consequences on survival, general growth and development and therefore are not considered to be adverse. There was no statistical significance and no indication of a test item-related trend in type or incidences of other skeletal findings and they were therefore considered to be spontaneous in nature. See table 6 for a summary.
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no serious toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature. There was no statistical significance observed in litter incidences of findings in treatment groups when compared with the controls and no indication of a test item-related trend in the type and incidences of visceral findings.

See table 6 for a summary.
Other effects:
no effects observed
Description (incidence and severity):
Craniofacial Examination:
Craniofacial examination by razor blade serial sectioning technique revealed no findings in all groups including control group.
Details on embryotoxic / teratogenic effects:
No indication of embryotoxic or teratogenic effects were observed up to highest dose tested.

For detailed information on prenatal/litter data, please refer to Table 4 in section "Any other information on results incl. tables".
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: based on number of terminally sacrificed females
Remarks on result:
other:
Remarks:
No indication of developmental or foetal toxicity was observed up to highest dose tested. However, based on number of terminally sacrificed females, MD (150 mg/kg bw) is considered as NOAEL for this study.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Dose Formulation Analysis:

The recoveries observed for the low dose (LD) group at 33.33 mg/mL were between 96.6 % and 102.1 %, for the medium dose (MD) group at 100.00 mg/mL were between 95.6 % and 101.3 %, and for high dose (HD) group at 216.67 mg/mL were between 99.8 % and 100.2 %. The mean recoveries observed in the LD, MD and HD groups were 100.2 %, 99.8 %, and 100.0 % of the nominal concentration, respectively. The recovery for HD-2 group at 166.67 mg/mL was 101.5 % and for LID group at 13.33 mg/mL was 96.3 %. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %.

Further Results:

Table 1. Summary Body Weight Changes (g)

Sex: Female - Phase: Gestation

Days  -  C/F  LD/F  MD/F  HD/F  LID/F
0–3

Mean

SD

N

75.4a

55.2

20 

82.7

68.3

17 

62.2

59.2

17 

66.0X

63.6

52.4

18 

3–6

Mean

SD

N

 21.8k

47.9

20

67.5

123.1

17 

25.1

35.9

17 

121.0X

46.3

64.0 

18

6–9

Mean

SD

N

-63.7u

79.7

20 

-139.9

126.5

17 

-208.5***

68.0

17 

-286.0X

-109.7

92.0

18 

9–12

Mean

SD

N

29.2 ad

83.5

20 

13.3

85.4

17 

-85.6***

67.0

17 

 -48.0X

1

25.4

77.1

18

12–15

Mean

SD

N

 14.7u

66.5

20

-48.2*

69.4

17

 16.9

115.2

17

79.0X

1

33.9

117.6

18

15–18

Mean

SD

N

-3.8a

80.5

20

-35.8

90.9

17

 31.1

122.0

17

185.0X

1

-21.4

105.7

18 

18–21

Mean

SD

N

 -5.5K

82.0

20

21.2

105.2

17

-16.5

86.0

17

72.0X

-26.8

80.1

18

21–24

Mean

SD

N

-9.9K

90.8

20

-37.5

71.2

17

-52.6

115.0

17

53.0X

1

0.3

67.9

18

24–27

Mean

SD

N

43.3u

82.4

19

-73.7**

113.0

15

-51.1*

134.2

17

 -10.0X

1

-6.1

116.1

15

0–27

Mean

SD

N

98.7u

223.4

19

-107.3*

226.5

15

-278.8***

358.9

17

 232.0X

1

20.7

284.3

15

d = day; a = ANOVA; X = Group excluded from statistics; k = KRUSKALL-WALLIS; u = KRUSKALL-WALLIS-DUNN; *** = p < 0.001; ad = ANOVA-DUNNETT; * = p < 0.05; ** = p < 0.01

Table 2. Summary Food Consumption (g/day/animal)

 Days  -  C/F  LD/F  MD/F  HD/F  LID/F
0–3

 Mean

SD

N

Deviation Vs Control (%)

165.5K

137.5

20

0.0

162.8

52.0

17

-1.6

147.0

61.6

17

-11.2

164.0

1

-0.9

148.6

33.8

18

-10.2

3–6

Mean

SD

Deviation Vs Control (%)

153.0K

50.9

20

0.0 

166.3

42.1

17

8.7

156.0

45.8

17

1.9

196.7X

1

22.1

156.5

37.4

18

2.3

6–9

Mean

SD

Deviation Vs Control (%)

65.5u

123.2

20

0.0

58.6

48.9

17

-71.3

18.8***

20.0

17

-71.3

7.7X

1

-91.2

43.7

34.8

18

-33.2

9–12

Mean

SD

Deviation Vs Control (%)

94.6u

48.2

20

0.0

76.1

53.1

17

-19.6

22.8***

26.4

17

-75.9

 48.0X

1

-49.3

76.7

50.2

18

-18.9

12–15

Mean

SD

Deviation Vs Control (%)

84.1K

129.7

20

0.0 

33.2

28.6

17

-60.5

43.6

47.7

17

-48.1

59.0X

1

-29.8

48.4

38.3

18

-42.4

15–18

Mean

SD

Deviation Vs Control (%)

73.1K

76.8

20

0.0 

36.3

45.5

17

-50.3

65.6

59.5

17

-10.2

110.3X

1

43.4

 60.9

45.5

18

-16.7

18–21

Mean

SD

Deviation Vs Control (%)

 69.2K

33.8

20

0.0

51.0

41.0

17

-26.2

75.6

50.4

17

9.3

91.0X

1

31.6

54.7

36.0

18

-20.9

21–24

Mean

SD

Deviation Vs Control (%)

55.8K

52.4

20

0.0

55.9

47.5

17

0.2

56.4

45.8

17

1.1

137.0X

1

112.9

64.5

62.4

18

15.6

24–27

Mean

SD

Deviation Vs Control (%)

72.7ad

35.5

20

0.0 

44.6

39.0

15

-38.6

42.4*

35.3

17

-41.6

81.7X

1

12.4

61.5

35.1

15

-15.3

Total

Total (g)

Mean (g)

833.5

92.6 

684.8

76.1

628.2

69.8

895.4

99.5

715.5

79.5

d = day; k = KRUSKALL-WALLIS; X = Group excluded from statistics; u = KRUSKALL-WALLIS-DUNN; *** = p < 0.001; ad = ANOVA-DUNNETT; * = p < 0.05

Table 3. Summary Fetus Weight (g)

Fetus Weight [g] 

 C / F LD / F   MD / F  HD / F  LID / F
 Male

Mean

SD

Deviation Vs Control (%)

33.54u

5.95

81

0.00

27.92***

6.58

56

-16.77

31.42

7.65

44

-6.31

32.80X

8.06

2

-2.21

30.75*

7.15

68

-8.32

Female

Mean

SD

Deviation Vs Control (%)

30.90u

5.83

88

0

27.25**

6.57

70

-11.82

29.38

8.12

53

-4.94

30.04

3.08

7

-2.78

31.49

7.34

52

1.89

u = KRUSKALL-WALLIS-DUNN; *** = p < 0.001; X = Group excluded from statistics; * = p < 0.05

Table 4. Summary Prenatal Data/Litter Data

C/F LD/F MD/F HD/F LID/F
Number of pregnant dams N 20 17 17 1 19
Number of pregnant dams, used for calculation N 20 16 17 1 18
Number of non-pregnant dams N 0 2 3 0 1
Early delivery N 0 0 0 0 0
Corpora Lutea Total 188 174 140 10 174
No. per animal Mean 9.4u 10.9 8.2 10.0X 9.7
S.D. 1.9 2.22 1.89 2.68
N 20 16 17 1 18
Implantation Sites Total 178 162 120 10 160
No. per animal Mean 8.9u 10.1 7.1 10.0X 8.9
S.D. 2.02 2.87 2.61 3.07
N 20 16 17 1 18
Preimplantation Loss Total 10 12 20 0 14
No. per animal Mean 0.5k 0.8 1.2 -X 0.8
S.D. 0.95 1.53 1.7 1.59
N 20 16 17 1 18
% per animal Mean 5.2k 7.4 15.1 -X 8.7
S.D. 10.03 15.26 24.1 18.17
N 20 16 17 1 18
Fetuses  Total 170 127 105 9 121
No. per animal Mean 8.5k 7.9 6.2 9.0X 6.7
S.D. 2.01 4.15 2.92 4.16
N 20 16 17 1 18
Alive % 99.4 99.2 92.4 100 99.2
Dead (stillbirths) % 0.6 0.8 7.6 0 0.8
Live Fetuses Total 169 126 97 9 120
No. per animal Mean 8.4k 7.9 5.7 9.0X 6.7
S.D. 2.09 4.06 3.24 4.17
N 20 16 17 1 18
Number of dams with dead fetuses N 1 1 1 0 1
Dead Fetuses Total 1 1 8 0 1
No. per animal Mean -k 0.1 0.5 -X 0.1
S.D. 0.22 0.25 1.94 0.24
N 20 16 17 1 18
% per animal Mean 0.8k 0.4 5.2 -X 0.6
S.D. 3.73 1.47 21.56 2.36
N 20 16 17 1 18
Number of dams with resorptions N 6 5 7 1 7
Early Resorption Total 5 8 7 1 7
No. per animal Mean 0.2k 0.5 0.4 1.0X 0.4
S.D. 0.55 0.82 0.62 0.61
N 20 16 17 1 18
% per animal Mean 2.5k 4.5 5.6 10.0X 5
S.D. 5.5 7.38 8.32 9.03
N 20 16 17 1 18
Late Resorption Total 3 8 1 0 7
No. per animal Mean 0.2k 0.5 0.1 -X 0.4
S.D. 0.49 0.89 0.24 0.85
N 20 16 17 1 18
% per animal Mean 1.8k 4.7 0.7 -X 3.8
S.D. 5.76 8.85 2.69 8.52
N 20 16 17 1 18
Number of dams with abortions N 0 3 1 0 1
Abortion Sites  Total 0 19 7 0 25
No. per animal Mean -k 1.2 0.4 -X 1.4
S.D. - 3.37 1.7 3.36
N 20 16 17 1 18
Postimplantation Loss Total 9 36 23 1 40
No. per animal Mean 0.4k 2.2 1.4 1.0X 2.2
S.D. 0.69 3.44 2.6 3.26
N 20 16 17 1 18
% per animal Mean 5.2k 22.1 17.4 10.0X 26.1
S.D. 7.74 34.29 32.12 36.77
N 20 16 17 1 18
Males Total 81 56 44 2 68
No. per animal Mean 4.0k 3.5 2.6 2.0X 3.8
S.D. 1.1 2.31 1.73 2.69
N 20 16 17 1 18
Females Total 88 70 53 7 52
No. per animal Mean 4.4k 4.4 3.1 7.0X 2.9
S.D. 1.9 2.5 1.93 2.27
N 20 16 17 1 18
% of Males per animal Mean 49.8a 43.3 43.5 22.2X 54.7
S.D. 14.44 15.59 19.87 22
N 20 14 15 1 15
Fetus weight (g) Mean 32.8a 28.3 32.3 30.7X 32.4
S.D. 4.54 4.54 7.81 6.97
N 20 14 15 1 15
Fetus weight of Male Fetuses (g) Mean 33.6a 29 32.6 32.8X 31.2
S.D. 5.2 4.8 8.1 6.2
N 20 14 14 1 14
Fetus weight of Female Fetuses (g) Mean 31.5a 28.1 31.8 30.0X 31.8
S.D. 4.23 4.91 7.97 8.39
N 20 14 15 1 15
u = KRUSKALL-WALLIS-DUNN; X = Group excluded from statistics; k = KRUSKALL-WALLIS; NA = No Test Applicable; a=ANOVA          
Preimplantation Loss = Corpora Lutea - Implantation Sites  
Postimplantation Loss = Early/Late resorptions + Aborted Fetuses + Dead Fetuses    
Affected Implants = Early/Late resorptions + Aborted Fetuses + Dead Fetuses + Malformed Fetuses        

Table 5. Summary mortality

 

 

Number/

mg/kg bw/d

C

0

 

LID

20

LD

50

MD

150

HD

325/250

Day 0-27

Animals examined

N

20

20

20

20

16

Animals with signs

N

0

1

1

0

15

Dead

N

0

1

1

0

15

Found dead

N

0

0

0

0

2

Euthanized

N

0

1

1

0

13

 

Table 6. Summary of Fetal malformations and variations (presented as no. of fetuses)

Doses [mg/kg bw/d]

C

0

LID

20

LD

50

MD

150

HD

325/250

External malformations (no. of fetuses) N 3 1 0 0 1
External variations N 0 0 0 0 0
Skeletal malformations N 0 0 0 0 0
Visceral malformations and variations N Incidences of a few malformation and variations were observed for various organs. However, these were not statistically significant from control or followed a dose-response.
Total Craniofacial Observation N 0 0 0 0 0
Skeletal variations
Vertebral sacral arches supernumerary ossification Litters affected N 11i 0 0 0 0
% 55 0 0 0 0
Fetuses affected N 24 0 0 0 0
% per litter Mean 13.9u  0*** 0*** 0*** 0
S.D. 18.54 0 0 0
Deviation vs. control 0 -100 -100 -100
Unossified 5th sternebra Litters affected N 7i 8 12* 9 1
% 35 53.3 85.7 60 100
Fetuses affected N 11 15 52 19 1
% per litter Mean 5.99u 12.22 40.99*** 15.21 11.11
S.D. 9.35 15.24 29.48 16.4
Deviation vs. control 0 104.1 584.7 154.1 -35
Unossified hindlimb talus Litters affected N 2c 4 7 5 0
% 10 26.7 50 33.3 0
Fetuses affected N 2 10 22 7 0
% per litter Mean 1.06u 8.36 13.39 5.77
S.D. 3.25 19.23 23.09 10.4
Deviation vs. control 0 692.5 1168.9 446.4

R= Region: Lower-Extremity (Bone), RLB = Region: Lower-Extremity (Bone), c=CHI-SQUARE, k=KRUSKALL-WALLIS, R = Region: Head-Neck Bone), RHB = Region: Head-Neck (Bone), u=KRUSKALL-WALLIS-DUNN, *** = p < 0.001, * = p < 0.05. R= Region: Trunk (Bone), RTB = Region: Trunk (Bone), i=CHl-SQUARE-FISHER

Conclusions:
In a prenatal developmental toxicity study conducted according to OECD testing guideline 414, the test item was prepared using sunflower oil as vehicle and orally administered to 20 female New Zealand White rabbits/dose group at dose levels of 0, 20 (LID), 50 (LD), 150 (MD) or 250/325 (HD) mg/kg bw/day from days 6 through 27 of gestation. Based on the results of the study, the NOAEL for maternal toxicity was considered to be 50 mg/kg bw/day. There was no indication of developmental or foetal toxicity observed up to the highest dose tested. However, based on number of terminally sacrificed females, the NOAEL for developmental toxicity is considered to be 150 mg/kg bw/day.
Executive summary:

In a prenatal developmental toxicity study conducted according to OECD testing guideline 414, Bis(a,a-dimethylbenzyl)peroxide (purity 99.9%) was administered to 20 female New Zealand White rabbits/dose by oral gavage at dose levels of 0, 20 (LID), 50 (LD), 150 (MD) or 250/325 (HD) mg/kg bw/day from days 6 through 27 of gestation. From treatment day 22, the dose level of HD group was reduced from 325 mg/kg bw to 250 mg/kg bw. On gestation day (GD) 28 the animals were sacrificed and examined.

No test item-related mortality, clinical signs, body weight, food consumption and caesarean parameter was observed in LID, LD and MD group of treated female rabbits during the treatment period.

Moribund sacrifice of one each female from LID and LD was attributed to a gavaging error. Furthermore, 2 mortalities and 13 morbidities from HD group were considered to be due to the severe local disturbance of the functioning of the gastro-intestinal tract at this dose level. The complete HD group (except 1 terminally sacrificed female) was sacrificed before scheduled terminal sacrifice. Also, all clinical signs observed in terminally sacrificed females up to MD group were incidental or non-adverse in nature. In addition, reduced food intake was observed in almost all animals including control group. In HD group females, in addition to reduced food intake, test item related reduced spontaneous activity was observed. Five females (3 from LD 1 from LID and 1 from MD) showed signs of abortion prior to the scheduled sacrifice. All these effects were considered to be attributed to the local disturbance caused by the test substance in the gastro-intestinal tract (irritation, osmosis, regurgitation, unabsorbed material) of rabbits by eating the soft faecal pellets leading to malnutrition, reduced spontaneous activity, loss of body weight and abortion. The gastro-intestinal tract of the rabbit is unique compared to other animals and has no relevance to humans.

No treatment-related effect on body weight, food consumption was observed in LID and LD group. There were effects observed on body weight and food consumption in MD group. It is well know that ingestion of organic peroxides (highly reactive substances) result in local gastrointestinal irritation leading to feed refusal and severe weight loss. Therefore, oral administration of the concentrated test item caused local irritation due to the disturbance of the gastric content preventing rabbits from eating leading to malnutrition and loss of body weight.

No treatment-related effect on prenatal data parameters, litter data parameters and gross pathology of terminally sacrificed females was observed at any dose tested. The findings from majority of dead or moribund sacrificed HD females at necropsy were considered due to the severe local disturbance of the functioning of the gastro-intestinal tract as discussed above. Furthermore, no treatment-related and toxicologically relevant external, visceral or craniofacial findings were observed in the treatment groups when compared with the controls. Based on the results in this prenatal developmental toxicity study in New Zealand White pregnant female rabbits with Bis(a,a-dimethylbenzyl)peroxide, the NOAEL for maternal toxicity in this study is considered to be 50 mg/kg bw/day. No indication of developmental or foetal toxicity was observed up to highest dose tested. However, based on number of terminally sacrificed females, the NOAEL for developmental toxicity is considered to be 150 mg/kg bw/day.

The developmental toxicity study in the rabbit is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rabbit. 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
chronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 414 (rat) 

In a developmental toxicity study according to OECD 414, dicumyl peroxide was administered to 24 pregnant female 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 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. The maternal LOAEL is 450 mg/kg bw/day. The maternal NOAEL is 150 mg/kg bw/day. 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 discolouration or fibrinoid degeneration.

 

Based on the initial study report it was not possible to clearly exclude any developmental effect. In 2015, an independent expert pathologist was re-analysing the stained skeletons. In this context a review from Kimmel et al. (2014, “Relationship Between Bent Long Bones, Bent Scapulae, and Wavy Ribs: Malformations or Variations?”) would need to be taken into account. The authors’ main conclusion is that “bent long bones and scapulae, like wavy ribs, appear to be secondary to maternal and developmental toxicity, are transient, and like wavy ribs should be considered variations rather than malformations.” It needs to be evaluated whether or not this conclusion holds also true for the effects reported here.

In the re-examination, except for occasional differences in data relating to the girdle and limb findings noted at the original examination, there were no additional skeletal malformations Also, observation of malrotated hindlimb in high dose foetuses at external examination did not always occur in individuals that were subsequently found to have malformed long bones. This is a condition which rarely results in any apparent skeletal abnormality and, as such, is not always classified as malformation. In foetuses which show no other major abnormality, it is usually associated with low foetal weight and/or poor general condition, possibly resulting in reduced in utero movement. The latter conditions are often related to maternal toxicity.

The data reported by the original examiners for the critical findings of wavy ribs, accompanied particularly at the high dose by bent pectoral girdle structures and long bones in fore and hindlimbs, were found to be generally reliable with one proviso regarding descriptive wording. Findings in the pectoral and long bones were frequently described as bent and “short” but it is considered that apparent shortening of these bones is usually a further consequence of the change in appearance due to bending, and not “shortening” per se. Thus, use of both words separately is potentially misleading and, if short is to be used at all, “bent/short” would be more appropriate terminology. There were some small differences in findings at the two examinations. At re-examination, some structures that had not been noted at the original examination were found to be abnormal and there were occasional cases where structures found to be abnormal at the first examination were not confirmed or considered to show a different severity grading at the re-examination. Severity gradings were somewhat inconsistent in respect of wavy ribs and since this has influenced the inclusion of data within the group incidence appendices, a full re-evaluation of the study could slightly change the result for these data. In terms of the more minor issues, the picture was more difficult. There were numerous small discrepancies in the ossification data, particularly affecting the findings for sternum, metatarsals and skull ossification and, in the case of the latter, also involving the severity gradings applied to the findings. However, as much of these data are not included in the group incidence table, which is assumed to be the basis of the analysis of results, the only reported parameter likely to be affected by this procedure is skull ossification.

The reported data for the critical findings of wavy ribs and bent/short pectoral and limb structures were found to be generally reliable. In terms of the study result, the high incidence of these findings at the high dose was of clear concern and the occurrence of three foetuses with bent scapula (malformation) in two litters at the medium dose was also of possible importance. There were no similar concerns in respect of two foetuses from one low dose litter with scapula bent slight, although it should be noted that one of these findings was considered, at re-examination, to show moderate severity, which would mean that the classification for this individual would change to malformation.

Possible etiology of findings

With regard to the formation of wavy ribs in the rat, it has been suggested by many authors [10][15] that a delay in ossification (i.e. lack of mineralisation) in the middle of the ribs, at around Day 17 of gestation, leaves them susceptible to bending due to the force of intrauterine pressure. It is of interest that lack of ossification in the middle of the ribs was clearly visible, at Day 20 of gestation, in at least one of the high dose foetuses in the Toxi-Coop study. With respect to long bones, a similar scenario is proposed in which bending is thought to be due to pressure exerted by the surrounding muscle [12]. If there is calcium deficit due to delayed ossification, the bones will bend particularly when they are also under pressure within the uterus. For the scapulae, the suggestion from the literature is that they are bent due to the stress from muscular contractions that occur during their calcification period which is much earlier (it is the second bone to begin ossification) than for many other bones. For the humerus, which tends to become thickened and misshapen in the region of the deltoid process (rather than bent), overproduction of osteoid is thought to be the cause. Recovery from these problems after birth is considered to be due to absence of stress when the animal is freed from the intrauterine environment. The developing bones can then remodel and proceed with normal development.

Findings of this type have also been described by some authors [19] as variable incidences of a “chondrodystrophy” syndrome. This syndrome, at worst, can involve multiple skeletal abnormalities, some of which are very severe (e.g. cleft palate and brachygnathia, spinal column malformations such as lordosis and kyphosis), as well as wavy ribs and bent girdle and limb bones but, more commonly, is restricted to wavy ribs and bent girdle and limb bones. It is believed to be caused by genetic mutations which affect the hyaline cartilage and it appears that some Wistar rats are predisposed to show these findings at a background incidence in controls and more frequently at higher incidences under the stress of test item investigations

Classification of critical findings

Classification of findings as malformations or variations is a system that was adopted to indicate the relative importance of findings. However, it can be the cause of unnecessary concern because the mention of malformation tends to be emotive. In studies of this type, the most important factor is usually the incidence of particular findings across the groups. If the incidence of finding is clearly increased in one or more treated groups, potential hazard is usually clear regardless of whether the findings are classified as malformations or variations.

The wavy ribs in this study, which occurred in increasing numbers in the medium and high dose groups, have been classified by the original examiners as variations and this is considered acceptable. The classification of wavy ribs (also called wavy/locally thickened or kinked in other laboratories) as variations, regardless of severity grading, is commonly accepted in most laboratories and, within the literature, there is much supporting evidence for transience of these effects [8][10][13][14].

Bent/short pectoral and limb bones occurred mainly at the high dose of this study but occasional affected scapulae were seen in other groups, including controls. These findings were classified by the original examiners as malformation or variation, dependent upon the severity of effect. Findings marked as “slight” were classified as variations; findings considered to be “moderate” (with no severity grading attached) were classified as malformations. This is also considered an acceptable procedure and is the current practice in other laboratories within Europe but it should be noted that, more recently, classification of such findings as malformations has come under some scrutiny [12].

It is possible that, even the more markedly affected girdle and limb bones, could be considered to be variations. The working definitions for malformation and variation are worded slightly differently in different laboratories but the key criteria of permanent or transient and harmful or not harmful are generally the same. Thus a malformation is typically described as “a permanent structural change, likely to adversely affect the survival or health of the individual” whilst a variation is “a change, usually transient in nature, that is unlikely to adversely affect survival or health of the individual”.

Investigation of the postnatal development of foetuses that have shown bent/short pectoral girdle and limb bones seems to suggest that even structures that have been severely affected in late gestation can be recovered soon after birth and that development thereafter is normal [8]. Most of this work has been done using littermates from dams treated at doses known to cause the effects and terminating them on different postnatal days. More recently, however, with use of micro CT scanning [8], it has been possible to investigate the same pups at multiple different postnatal ages.Thus, it appears 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.

The classification change discussed above has recently been proposed by very well respected scientists from within the industry [12]. In practice, however, the new ideas could seem to be very contentious. For foetal examiners, who see the extent of the changes, the new thinking, though logical, is likely to be difficult to accept, particularly in a case such as the current study, where it would mean that the decisions of the original examiners might need to be overruled. This might also be true for reviewers and regulators. Given the nature of the bending of girdle and limb bones, in the majority of affected fetuses in the high dose of this study and in many other studies that are affected by these changes, it can appear unrealistic to conclude that a resultant disadvantage to the affected animal during the critical early neonatal period of its life has no long-term consequences for later life. Moreover, although the foetuses in this study showed very few external changes and no visceral changes that were considered to be related to the test item, there could be other, as yet, unknown physical or biochemical effects associated with these findings. There is a large number of different compounds that have elicited findings of this type and so many different biological systems could be involved [10][11][12][16]. Looking at the issue from this perspective, it would appear that, so far, the evidence for change is by no means conclusive for all possible mechanisms that could lead to the girdle and limb changes that have been observed.

As a conclusion, the definition of the NOAEL at 150 mg/kg could be seen as a conservative approach in the absence of exhaustive toxicological and mode of action data which would confirm the post-natal reversibility of the findings and would allow setting the NOAEL at 450 mg/kg. However, the fact that the findings might not only be reversible but also of short duration and not harmful to the animal in the longer term, classification as Repr. 1B is not warranted.

Maternal Toxicity

In this study, the presence of maternal toxicity at the high dose, where the majority of the bent/short girdle and limb changes occurred, is important. It suggests that the foetal effects are secondary to maternal effects and not, therefore, a selective effect of the test item on the foetus (i.e.not teratogenicityper se). This idea is further supported by evidence in the literature which shows that findings of this type are usually accompanied by maternal toxicity and thus thought to be the result maternal stress [7][11][12]. Also, from the literature, it is evident that there is a range of different types of compound that are known elicit this type of finding, particularly in the treated groups where maternal stress is most likely. A variety of classes of agents with differing modes of action have been shown to produce effects on long bones and scapulae as well as wavy ribs [12] and so it is clear that the findings in this study are not unique to this test item. It is considered that the opinion of the original examiners that the changes seen at the high dose in this study are likely to be related to maternal toxicity, is plausible. The group values in the current study clearly demonstrate maternal toxicity. Clinical signs, reduced body weight change (including a small amount of absolute body weight loss) and reduced food intake, were observed in the high dose group and there was also reduced body weight gain at the medium dose.

The position relating to the medium dose

If the classification of foetal findings remains as suggested by the original examiners, the NOAEL for developmental toxicity, which is set at the medium dose of 150 mg/kg, is a subject for discussion. In this group there were three incidences of bent scapula (severity moderate and therefore classified as malformation) in two litters with no accompanying limb malformations and these findings occurred in the presence of reduced maternal body weight gain. In setting the NOAEL at this dose level, it was judged by the original examiners that the three bent scapulae are within the background range. However, in the light of the result at the high dose, this judgement will need to be robustly justified. This incidence of bent scapula is low and, in the absence of comparable current control values, the best way to defend such data is usually by use of historical data. In this case, however, it is considered that the historical data, as currently presented, are not detailed sufficiently robust to reassure that the malformed scapulae are not test item-related. However, based on the overall result of the re-evaluation this seems to be a reporting issue and does not generally disqualify the conclusion drawn by the study author.

 

[7] Carney EW, Kimmel CA. 2007. Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs. Birth Defects Res B Dev Reprod Toxicol 80(6):473–496.

[8] De Schaepdrijver L, Delille P, Geys H, Boehringer-Shahidi C, Vanhove C. 2014. In vivo longitudinal micro-CT study of bent long limb bones in rat offspring.Reprod Toxicol 46:91–97.

[10] Kast A. 1994. “Wavy ribs:” a reverisble pathologic finding in rat fetuses.Exp Toxicol Pathol 46:203–210.

[11] Khera, KS. 1981. Common fetal aberrations and their teratologic significance:A review.Fundam Appl Pharmacol 45: 435–444.

[12] Kimmel CA, Garry MR, DeSesso JM. 2014. Relationship between bent long bones, bent scapulae, and wavy ribs: malformations or variations?Birth Defects Res B Dev Reprod Toxicol 101:379-392.

[13] Mitchard TL, French J. Apparent postnatal recovery of chondrodystrophy in theHarlan Han Wistart rat. Reprod Toxicol 2011;32:169–70.

[14] Mitchard T, Stewart J. 2014. Reduced post-natal versus pre-natal incidence of bent long bones and scapulae in a preliminary investigation using the Han Wistar rat.Reprod Toxicol 45:39–44.

[15] Nakatsuka T. 1988. Role of myometrial constriction in the induction of wavy ribs in rat fetuses.Teratology 37(4):329–334.

[16] Sterz H, Sponer G, Neubert P, Hebold G. 1985.A postulated mechanism of beta- sympathomimetic induction of rib and limb anomalies in rat fetuses.Teratology 31(3):401–412.

[19] Wilby O, Critchell K, Coulby DA. Chondrodystrophy syndrome in the laboratory rat.Reprod Toxicol 2007; 24(91):57.

 

OECD 414 (rabbit)

 

In a developmental toxicity study Bis(a,a-dimethylbenzyl)peroxide was administered to 20 female New Zealand White rabbits/dose by gavage at dose levels of 0, 20 (LID), 50 (LD), 150 (MD) and 325/250 (HD) mg/kg bw/day from days 6 through 27 of gestation.

There were no test item-related effects of toxicological relevance observed for the mean foetus weight, male and female foetus weight on litter basis (group mean of individual litter mean) in any of the treatment groups when compared with the controls.

Foetal external examination on the day of terminal sacrifice revealed no test item related external findings in foetuses of any of the treated groups. Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no serious toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature. There was no statistical significance observed in litter incidences of findings in treatment groups when compared with the controls.

Craniofacial examination by razor blade serial sectioning technique revealed no findings in all groups including control group. Skeletal examination of the foetuses after the Alizarin red staining technique for skeletal findings revealed a range of findings which were of a type or which occurred at an incidence generally comparable to or slightly lower or higher in the dose groups when compared to the control group.

No test item related gross pathological changes of toxicological significance were observed during the macroscopic examination of the terminally sacrificed females of the control, LD, MD and HD groups. At macroscopic evaluation of the majority of dead or moribund sacrificed HD females at necropsy, major findings observed were fluid filled thoracic cavity, dark fluid filled and abnormal lung colour, gas filled colon, enlarged and abnormal coloured gallbladder which were considered were considered due to the severe local disturbance of the functioning of the gastro-intestinal tract. No indication of developmental or foetal toxicity was observed up to highest dose tested. However, based on number of terminally sacrificed females, HD (325/250 mg/kg bw) did not provide enough offspring for a statistical assessment. For practical reasons, MD (150 mg/kg bw) is considered as NOAEL for this study (in absence of adverse effects in HD).

Justification for classification or non-classification

Based on the initial OECD 414 in rat it was not possible to clearly exclude developmental effects. Foetal malformations were found at the highest dose level (450 mg/kg bw/d), which is also the LOAEL for maternal toxicity.

The Norwegian Environment Agency concluded in the CHL report for Bis(α,α-dimethylbenzyl) peroxide that in the OECD 414 in rat it cannot be “unequivocally demonstrated on a case-by-case basis that the developmental effects are secondary to maternal toxicity as it should be according to the CLP guidance in order to not be classified”. The agency considered the developmental toxicity to be independent of the maternal toxicity and not to be a secondary non-specific consequence of the other toxic effects. And further, that according to the CLP guidance, even developmental effects occurring together with maternal toxicity can be the base for classification: “classification shall be considered where there is a significant toxic effect in the offspring, e.g. irreversible effects such as structural malformations, embryo/foetal lethality, significant postnatal functional deficiencies" which is the case for dicumyl peroxide as a higher incidence of malformations and embry/foetal lethality was seen in the high dose group.

However, the recommendation of the Norwegian Environment Agency was based on limited data and consists of one prenatal developmental toxicity study in rats, only. The findings of developmental toxicity, “although clear”, are limited to the high dose group and no clear dose-response is observed over the range of the three dose groups. The Agency therefore concluded, that “the severity and incidence of developmental toxicity in this study may not be enough to warrant classification in category Repr. 1B. However, the findings justify classification in at least category Repr. 2, as evidence of developmental the pups cannot unequivocally be ascribed to the effects seen in the dams”. At the 45 RAC meeting, the Norwegian Environment Agency proposal to classify bis(a,a-dimethylbenzyl)peroxide for Repr. 2 (H361d) was discussed. RAC was of the opinion, that “clear effects of reproductive toxicity, such as increased malformations do not correlate with maternal toxicity, and that these effects would be sufficient to propose a CLP classification as Repr. 1B (H360D)”.

 

The registrants agree that it is not possible in the OECD 414 in rat to unequivocally demonstrated on a case-by-case basis that the developmental effects are secondary to maternal toxicity. However, because of deficient historical control data it could also not be unequivocally demonstrated that effects were adverse, i.e. not within historical controls. In addition, during re-evaluation of the findings by an independent expert pathologist, it was concluded, that the critical findings of wavy ribs, accompanied particularly at the high dose by bent pectoral girdle structures and long bones in fore and hindlimbs 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.

If the OECD 414 in rat would be the only available study, the findings could justify classification in category Repr. 2 as proposed by the Norwegian Environment Agency - as evidence of developmental effects in the pubs cannot unequivocally be ascribed to the effects seen in the dams. However, new data from a developmental toxicity study in the second species (OECD 414, rabbit), where no indication of developmental or foetal toxicity was observed up to highest dose tested, do not support the interpretation of inconclusive effects in the rat study at high doses. Classification as toxic to reproduction is therefore not warranted.