Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 07 January 2016 Experimental Completion Date: 27 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Bisisobutyryl peroxide
EC Number:
222-340-0
EC Name:
Bisisobutyryl peroxide
Cas Number:
3437-84-1
Molecular formula:
C8H14O4
IUPAC Name:
2-methylpropanoyl 2-methylpropaneperoxoate
Details on test material:
- Name of test material (as cited in study report): Bisisobutyryl peroxide solution
- Substance type: Clear colourless liquid
- Physical state: Liquid
- Storage condition of test material: In freezer (≤ -25°C) in the dark


Specific details on test material used for the study:
Identification : Bisisobutyryl peroxide (CAS# 3437-84-1)
Physical State/Appearance : Clear colorless liquid
Purity : Diisobutyryl peroxide (38.91%)
Batch Number : BYK004856-171
Date Received : 25 November 2015
Storage Conditions : Approximately -20 °C
Expiry Date : 30 April 2016

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Animal Information
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 181 to 276g.

Animal Care and Husbandry
The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were
allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets.

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Arachis oil. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services. Results are given in Annex 1 and show the formulations to be stable for at least 4 hours when kept on crushed ice. Formulations were therefore prepared and kept on crushed ice before and during use.

Samples were taken of each test item formulation on two occasions during the study and were analyzed for concentration of Bisisobutyryl peroxide (CAS# 3437-84-1) at Envigo Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 79% to 104% of the nominal concentration; see Deviations from Study Plan.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preparation of calibration standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.1 mg/mL.

On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Calibration solutions were injected into the instrument, at the beginning and end of each sample analysis sequence as a minimum.

Preparation of test samples
The formulations received were kept on ice and extracted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent. This was then skaen to extract. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

The concentration of test item in the final solution was quantified by HPLC using UV detetection as detailed in the instrument parameters detailed below.

HPLC: Agilent Technologies 1200, incorporating autosampler and work station
Column: Luna 5 µ C18 (150 x 2 mm id)
Column temperature: 7°C
Autosampler temperature: 5°C
Mobile phase: acetonitrile:water (70:30 v/v)
Flor-rate: 0.4 mL/min
UV detector wavelength: 230 nm
Injection volume: 5 µL
Retention time: ~ 2.5 minutes

Details on mating procedure:
Not detailed in report
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
as two females from this group were found dead on the day after receiving their first dose, this dose level was deemed to be too high for continued dosing and was reduced to 600 mg/kg bw/day.
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
Another two females from this group were killed in extremis on Days 8 and 11 of gestation and the dose level was further reduced to 450 mg/kg bw/day
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
twenty-four time-mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats
Control animals:
yes, concurrent vehicle
Details on study design:
The starting dose levels and dose volume were selected in collaboration with the Sponsor based on available toxicity data including a 28 Day toxicity study in the rat (Notox Project: 498267). In the 28 day study, dose levels up to 1000 mg/kg/day did not result in any toxicologically relevant effects on body weight development or food consumption for animals of either sex. Treatment at 300 or 1000 mg/kg/day resulted in histopathological changes in the forestomach in both sexes that were considered to be reflective of irritant properties of the test item. Diffuse hepatocellular hypertrophy, associated with increased liver weights, was also observed in the liver of both sexes receiving 1000 mg/kg/day and was considered to be indicative of adaptive hepatic changes. Based on these results, a dose level of 1000 mg/kg bw/day was considered to be a suitable high dose for investigation in the present study together with 100 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Examinations

Maternal examinations:
Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each surviving individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.
Ovaries and uterine content:
The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.
Fetal examinations:
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):
Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16
V = viable fetus

The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
Percentage pre-implantation loss was calculated as:

((Number of corpora lutea - number of implantations) / number of corpora lutea) x 100

Percentage post-implantation loss was calculated as:

((Number of implantations - number of live foetuses) / number of implantations) x 100

Sex Ratio
Sex ratio was calculated as:

% male fetuses (sex ratio) = (Number of male fetuses / Total number of fetuses) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Of the females surviving to Day 20 of gestation, isolated instances of noisy respiration were observed for two animals from the high dose group with four females treated with 300 mg/kg bw/day also showing signs of respiratory distress on some days. Such clinical signs were not observed for any of the females given 100 mg/kg bw/day. Isolated instances of increased post-dose salivation were also detected for individual females receiving the test item at all doses but there was no dose-relationship.
One female from the 300 mg/kg bw/day dose group showed signs of hunched posture between Days 1 to 4 of gestation. As this was observed before dosing commenced, it was unrelated to treatment with the test item. One female receiving 100 mg/kg bw/day showed signs of fur loss towards the end of the dosing period. In the absence of similar findings in the remaining dose groups treated with the test item, this observation was deemed likely to be incidental.
Mortality:
mortality observed, treatment-related
Description (incidence):
In total, there were seven unscheduled deaths in the high dose group (1000/600/450 mg/kg bw/day) with another four in the intermediate dose group (300 mg/kg bw/day). - Please see below in "Any other information" for further details

With the exception of two females from the high dose group, which were found dead on Day 6 of gestation after receiving their first dose at 1000 mg/kg bw/day on the previous day and did not show any clinical signs before death, the remaining five premature decedents from this group were killed in extremis between Days 8 and 19 of gestation. This dose level was initially reduced to 600 mg/kg bw/day after females from this group had received one to three doses each followed by further reduction to 450 mg/kg bw/day later during the treatment period (see Text table in Section 3.1 for details). All animals killed in extremis showed signs of respiratory distress with individuals also exhibiting clinical signs of hunched posture, lethargy, piloerection, ptosis, hypothermia, distended abdomen and/or increased salivation. Most of the early decedents were observed with actual body weight losses and marked reductions in dietary intake prior to death. At necropsy, macroscopic findings for these animals included thickened glandular and/or non-glandular region/s in the stomach, raised limiting ridge, sloughing of the non-glandular region in the stomach and/or gaseous distension of one or more region/s in the gastrointestinal tract.
At 300 mg/kg bw/day, there were four unscheduled deaths between Days 14 to 19 of gestation with two animals found dead and the other two killed in extremis due to the severity of clinical signs. As with the high dose group, most of these premature decedents showed clinical signs of respiratory distress before death with staining around the snout and increased salivation also recorded for some individuals. Actual body weight losses and lower food consumption were also apparent for some of these animals whilst macroscopic findings included gaseous distension in the stomach or lower parts of the gastrointestinal tract. In general clinical signs and effects on body weight or food consumption in these animals appeared to be less severe than those observed for the early decedents in the high dose group.
There were no unscheduled deaths amongst the low dose (100 mg/kg bw/day) or control animals.
The clinical observations and macroscopic findings for the early decedents will not be further discussed in this section of the study report.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At the start of dosing (over Days 5 to 6 of gestation), group mean body weight loss was evident in the surviving high dose females (all dosed at 1000 mg/kg bw/day on Day 5 of gestation) but the difference from controls was not statistically significant. Recovery was evident thereafter and subsequent group mean body weight gains were similar to controls. Due to the initial effect on body weight development, however, cumulative body weight gains up to Day 8 of gestation were slightly lower than controls in these females (without attaining statistical significance) but overall body weight gain was similar to controls. The surviving females treated with 300 or 100 mg/kg bw/day did not show any effect of treatment on body weight performance throughout the test item administration period.
At all dose levels, group mean gravid uterus weights in the surviving females treated with the test item were similar to controls. The mean body weights and body weight gains, when adjusted for the contribution of gravid uterus weight, were also comparable with controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, the surviving high dose females showed slightly but statistically significantly lower dietary intake over Days 5 to 8 of gestation (p<0.01) with subsequent recovery evident. The corresponding values for the surviving females treated with 300 or 100 mg/kg bw/day were comparable with controls throughout the treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic findings at necropsy for any of the females surviving to Day 20 of gestation.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The following assessment is based on the 24, 24, 19 and 17 females with live young on Day 20 of gestation at 0 (control), 100, 300 and 1000/600/450 mg/kg bw/day, respectively. Female 69 from the 300 mg/kg bw/day was found not to be pregnant. This was an isolated event considered unrelated to treatment. All early decedents were confirmed to be pregnant (at necropsy).
There was no effect of maternal treatment on litter data as assessed by the mean number of implantations, in-utero offspring survival (as assessed by the mean number of early or late resorptions), live litter size, sex ratio and post-implantation losses at 100, 300 or 450 mg/kg bw/day. Pre-implantation losses and sex ratios across all test item-treated groups were also similar to controls. Additionally, fetal, litter or placental weights remained unaffected by treatment with the test item at all dose levels.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Please see "Any other information" for further details

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity.
Remarks on result:
other: Maternal toxicity

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
For all dose groups, there were no treatment-related trends in the proportion of fetuses (or litters) with evidence of external or visceral abnormalities.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant decrease in incidence in fetuses from the high dose group with incomplete ossification of the hyoid (p<0.01) was considered unlikely to be a consequence of treatment as this reflects improved hyoid ossification rates when compared to control group fetuses. A decrease in incidence in fetuses from all test item-treated dose groups with incomplete ossification of the pubis was also deemed unlikely to be a consequence of treatment as this reflects improved pubic ossification rates when compared to control group fetuses. The statistically significant difference at the low dose only (p<0.05) reflects the nature/limitations of the statistical methods employed. The increased incidence of ossification in the 1st vertebra was statistically significant at the low dose only (p<0.01). The lack of significant differences in incidence at both the intermediate and high dose levels shows that this was unlikely to be a treatment-related difference and more likely an example of normal biological variation.
Visceral malformations:
no effects observed
Description (incidence and severity):
For all dose groups, there were no treatment-related trends in the proportion of fetuses (or litters) with evidence of external or visceral abnormalities.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
skeletal malformations

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
450 mg/kg bw/day (actual dose received)
Treatment related:
no
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

In total, there were seven unscheduled deaths in the high dose group (1000/600/450 mg/kg bw/day) with another four in the intermediate dose group (300 mg/kg bw/day). Clinical signs before death and macroscopic findings for these early decedents were as following:

Summary of Mortalities for Study

Dose Level (mg/kg bw/day)

Animal Number

Day (Mode) of Death

Clinical Signs Prior to Death

Macroscopic Findings

300

49

19 (FD)

No abnormalities detected

Stomach: Gaseous distension

56

19 (KIE)

Noisy, reduced, labored, gasping respiration, staining around the snout

Stomach: Gaseous distension

62

14 (FD)

Noisy respiration

Duodenum, Jejunum, Ileum: Gaseous distension

64

18 (KIE)

Noisy, reduced, labored, gasping respiration, increased salivation

Stomach: Gaseous distension

1000/600/450#

75

17 (KIE)

Reduced, labored, gasping respiration, lethargy, distended abdomen

Gastrointestinal Tract: Gaseous distension

Ileum, Cecum: Yellow fluid

76

11 (KIE)

Reduced, labored respiration, hunched posture, lethargy, increased salivation

Stomach: Thickened glandular region, raised limiting ridge

Stomach, Duodenum, Jejunum, Ileum, Cecum: Gaseous distension

80

12 (KIE)

Reduced, labored respiration, hunched posture, piloerection, hypothermia

Stomach: Thickened glandular/non-glandular regions, raised limiting ridge

Stomach, Duodenum, Jejunum, Ileum, Cecum: Gaseous distension

88

19 (KIE)

Noisy, reduced, labored, gasping respiration, lethargy, ptsosis, increased salivation

Stomach: Gaseous distension

90

6 (FD)

No abnormalities detected

Stomach: Thickened glandular region

Stomach, Duodenum, Jejunum, Ileum: Gaseous distension

91

8 (KIE)

Noisy, labored, gasping respiration, increased salivation

Stomach: Thickened glandular/non‑glandular regions, raised limiting ridge, sloughing of the non-glandular region

Stomach, Duodenum, Jejunum: Gaseous distension

92

6 (FD)

No abnormalities detected

Stomach, Duodenum, Jejunum, Ileum, Cecum: Gaseous distension

 

#The starting dose level of 1000 mg/kg bw/day was initially reduced to 600 mg/kg bw/day and then further reduced to 450 mg/kg bw/day (see Text Table in Section 3.1 for details).

Females 56, 64, 88 and 91 were dosed on the day of death. The remaining early decedents received their last dose on the day prior to death.

FD = Found dead

KIE = Killedin extremis

Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

100

300

1000/600/450

Number of Fetuses (litters) Examined

167 (24)

157 (24)

135 (19)

113 (17)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

SKULL

 

 

 

 

 

 

 

 

 

 

 

 

Fontanelle (anterior) - large

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.7

Nasal - incomplete ossification

5

4

3.6

8

7

6.3

3

3

2.3

2

2

1.6

Frontal - incomplete ossification

2

2

1.2

2

2

1.2

0

0

0.0

3

3

2.8

Frontal - unossified area

2

2

1.1

0

0

0.0

0

0

0.0

0

0

0.0

Parietal - incomplete ossification

2

2

1.6

2

2

1.2

0

0

0.0

1

1

1.0

Parietal - unossified area(s)

1

1

0.5

0

0

0.0

0

0

0.0

0

0

0.0

Interparietal - incomplete ossification

8

6

5.1

11

8

7.4

8

4

5.8

3

3

2.4

Interparietal - unossified area(s)

0

0

0.0

0

0

0.0

2

2

1.4

0

0

0.0

Occipital (Supra-occipital) - incomplete ossification

5

4

2.9

5

4

3.2

3

3

2.2

2

2

1.9

Occipital (Supra-occipital) - unossified area(s)

5

4

3.2

8

6

6.5

3

1

2.0

3

3

2.6

Squamosal - incomplete ossification

11

8

6.7

8

8

5.2

5

4

3.8

7

6

6.4

Squamosal - unossified area(s)

1

1

0.5

1

1

0.5

0

0

0.0

4

3

3.6

Jugal - incomplete ossification

2

2

1.1

0

0

0.0

0

0

0.0

1

1

1.0

Zygomatic process of maxilla - incomplete ossification

10

7

6.3

6

5

3.7

8

5

6.0

4

2

3.9

Zygomatic process of squamosal - incomplete ossification

2

2

1.2

0

0

0.0

1

1

0.8

2

1

2.0

a


                                                Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

100

300

1000/600/450

Number of Fetuses (litters) Examined

167 (24)

157 (24)

135 (19)

113 (17)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

SKULL (continued)

 

 

 

 

 

 

 

 

 

 

 

 

Premaxilla - incomplete ossification

0

0

0.0

0

0

0.0

0

0

0.0

1

1

1.0

Hyoid - incomplete ossification

14

11

8.5

8

5

4.8

7

3

5.3

0

0

0.0**

Hyoid - not ossified

4

4

2.4

6

4

4.0

11

7

8.3

1

1

0.7

Tympanic annulus - incomplete ossification

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.7

Presphenoid - incomplete ossification

0

0

0.0

0

0

0.0

2

1

1.5

1

1

0.7

Basisphenoid - incomplete ossification

0

0

0.0

0

0

0.0

1

1

0.9

0

0

0.0

VERTEBRAL COLUMN

 

 

 

 

 

 

 

 

 

 

 

 

Odontoid - ossification present

0

0

0.0

1

1

0.8

0

0

0.0

0

0

0.0

Ventral arch of vertebra 1 - ossification present

29

15

18.5

57

19

36.9**

30

14

22.9

22

11

19.6

Cervical (neural) arch - incomplete ossification

3

2

1.5

0

0

0.0

1

1

0.8

2

2

1.6

Thoracic centrum - incomplete ossification

2

2

1.2

1

1

0.6

4

3

3.1

3

3

3.0

Thoracic centrum - not ossified

0

0

0.0

0

0

0.0

2

2

1.4

0

0

0.0

Thoracic centrum - bipartite ossification

0

0

0.0

1

1

0.6

2

2

1.6

1

1

1.2

Thoracic centrum - dumb-bell-shaped

10

7

6.4

10

7

6.6

11

6

7.9

12

6

10.9

a


 

Table11(continued)           Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

100

300

1000/600/450

Number of Fetuses (litters) Examined

167 (24)

157 (24)

135 (19)

113 (17)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

VERTEBRAL COLUMN (continued)

 

 

 

 

 

 

 

 

 

 

 

 

Thoracic centrum - asymmetrically ossified

1

1

0.6

1

1

0.5

2

1

2.1

1

1

0.8

Sacral (neural) arch - incomplete ossification

14

9

8.5

6

4

3.6

2

2

1.5

2

2

1.7

Sacral (neural) arch - not ossified

1

1

0.6

1

1

0.6

0

0

0.0

1

1

0.7

Caudal vertebrae - less than 4 ossified

32

14

19.0

29

11

18.6

28

9

20.5

14

7

13.6

Number of pre-sacral vertebrae = 25/27

2

1

1.0

1

1

0.6

0

0

0.0

2

1

1.5

RIBS

 

 

 

 

 

 

 

 

 

 

 

 

Ossification centre - associated with 1st lumbar vertebra

10

7

6.0

1

1

2.1

9

6

6.6

4

3

3.3

One or more ribs - thickened

1

1

0.7

0

0

0.0

0

0

0.0

0

0

0.0

Rib - short

2

2

1.1

1

1

0.6

0

0

0.0

1

1

0.7

Rib - rudimentary

1

1

0.5

1

1

0.6

0

0

0.0

1

1

0.7

Costal cartilage - misaligned

7

5

4.3

2

2

2.7

3

2

1.9

2

2

1.8

Costal cartilage - not fused to sternebra

14

10

8.7

7

7

4.5

6

5

4.3

8

5

7.2

a

 


 

Table11(continued)           Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

100

300

1000/600/450

Number of Fetuses (litters) Examined

167 (24)

157 (24)

135 (19)

113 (17)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

STERNEBRAE

 

 

 

 

 

 

 

 

 

 

 

 

Sternebra - incomplete ossification

3

3

1.6

0

0

0.0

1

1

0.8

1

1

1.0

Sternebra - not ossified

1

1

0.5

0

0

0.0

0

0

0.0

0

0

0.0

Sternebra - bipartite ossification

1

1

0.5

0

0

0.0

0

0

0.0

0

0

0.0

Sternebra - misaligned

6

5

3.4

1

1

0.6

5

4

3.4

3

3

3.0

Xiphoid cartilage - partially split

14

11

8.9

11

10

6.5

9

7

6.8

10

6

8.4

PECTORAL GIRDLE

 

 

 

 

 

 

 

 

 

 

 

 

Scapula - misshapen (comment on region)

2

2

1.3

4

2

3.5

2

2

1.6

2

2

1.8

PELVIC GIRDLE

 

 

 

 

 

 

 

 

 

 

 

 

Ischium - incomplete ossification

1

1

0.7

0

0

0.0

0

0

0.0

0

0

0.0

Pubis - incomplete ossification

4

4

2.4

0

0

0.0*

0

0

0.0

0

0

0.0

FORELIMBS

 

 

 

 

 

 

 

 

 

 

 

 

Metacarpal - not ossified

32

16

19.1

28

13

17.2

19

9

13.9

17

9

16.3

Forepaw phalanges - 1 or more - ossified

34

15

20.2

37

13

23.2

38

15

28.1

29

13

25.2

Humerus - incomplete ossification

3

3

1.8

0

0

0.0

0

0

0.0

0

0

0.0

Humerus - hole

2

2

1.3

0

0

0.0

0

0

0.0

1

1

0.7

a

 


 

Table11(continued)           Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

100

300

1000/600/450

Number of Fetuses (litters) Examined

167 (24)

157 (24)

135 (19)

113 (17)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

HINDLIMBS

 

 

 

 

 

 

 

 

 

 

 

 

Metatarsal - 1st - ossified

0

0

0.0

6

2

3.2

4

3

3.1

4

1

3.4

Metatarsal - incomplete ossification

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.7

Femur - incomplete ossification

8

6

5.2

3

3

1.9

0

0

0.0

2

2

1.7

Total

133

24

79.8

127

24

81.9

112

19

82.7

78

17

69.6

a


NOTE: afetus may appear in more than one category

Applicant's summary and conclusion

Conclusions:
The oral administration of Bisisobutyryl peroxide (CAS# 3437-84-1) to pregnant rats by gavage during gestation Days 5 to 19, at a dose level of 1000 mg/kg bw/day (first reduced to 600 mg/kg bw/day and then further reduced to 450 mg/kg bw/day) resulted in premature deaths of seven females between Days 6 and 19 of gestation. At 300 mg/kg bw/day, four females were found dead or killed in extremis between Days 14 and 19 of gestation. These deaths were considered to be related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. There were no unscheduled deaths amongst females treated with 100 mg/kg bw/day.

The surviving females from the high dose group showed lower body weight gain at the start of dosing, which was associated with slightly lower dietary intake. Recovery was evident thereafter and this initial effect was considered unlikely to be of any toxicological relevance. Excluding these early deaths from the overall assessment, 450 mg/kg bw/day could be established as the ‘No Observed Adverse Effect Level’ (NOAEL) for maternal systemic toxicity and the ‘No Observed Effect Level’ (NOEL) for developmental toxicity within the confines of this study.
Executive summary:

Introduction

The study was performed according to the study plan and was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study was designed to comply with the following guidelines:

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods

The test item was administered once daily by gavage to three groups, each of twenty-four time-mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats from Day 5 to Day 19 of gestation, inclusive. The starting dose level for the high dose group was 1000 mg/kg bw/day; however, as two females from this group were found dead on the day after receiving their first dose, this dose level was deemed to be too high for continued dosing and was reduced to 600 mg/kg bw/day. Another two females from this group were killedin extremison Days 8 and 11 of gestation and the dose level was further reduced to 450 mg/kg bw/day (see Text Table in Section 3.1 for details). Females from the intermediate and low dose groups were administered with 300 and 100 mg/kg bw/day, respectively, throughout the treatment period. A further group of twenty-four time-mated females was dosed with vehicle alone (Arachis oil) over the same treatment period to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

All surviving females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Mortality

In total, there were seven unscheduled deaths in the high dose group. Two females were found dead on Day 6 of gestation after receiving their first dose at 1000 mg/kg bw/day on the previous day and this dose level was reduced to 600 mg/kg bw/day. Another two females from this group were killedin extremison Days 8 and 11 of gestation and the dose level was further reduced to 450 mg/kg bw/day. Over Days 12 to 19 of gestation, a further three females from this dose group were killedin extremis. With the exception of the two females found dead on Day 6 of gestation, clinical observations of respiratory distress were evident in the remaining five early decedents with some of these also showing signs of hunched posture, lethargy, piloerection, ptosis, hypothermia, distended abdomen and/or increased salivation. In addition, most of these early decedents showed appreciable body weight losses and marked reductions in dietary intake prior to death. Macroscopic findings at necropsy consisted of thickened glandular and/or non-glandular region/s in the stomach, raised limiting ridge, sloughing of the non-glandular region of the stomach and/or gaseous distension of one or more region/s in the gastrointestinal tract.

At 300 mg/kg bw/day, two females were found dead on Days 14 and 19 of gestation and another two were killedin extremison Days 18 and 19 of gestation. As with the high dose group, most of these premature decedents showed clinical signs of respiratory distress before death with staining around the snout and increased salivation also seen in some instances. Body weight losses and slight decrease in food consumption were also apparent for some of these animals but the overall effects generally appeared to be of less prominence than those observed for the early decedents in the high dose group. At necropsy, macroscopic findings for these animals were confined to gaseous distension in the stomach or small intestine.

These early deaths were considered to be related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity.

There were no unscheduled deaths amongst females treated with 100 mg/kg bw/day.

Clinical Observations

Of the females surviving to Day 20 of gestation, isolated instances of respiratory distress were noted in individuals from the intermediate and high dose groups. A small number of females from all test item-treated dose groups also exhibited sporadic instances of increased post-dose salivation but there was no dose-dependence. These observations were deemed to be reflective of the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity.

Body Weight

For the surviving high dose females (all dosed at 1000 mg/kg bw/day), a slight decrease in body weight gain at the start of dosing (over Days 5 to 6 of gestation) was evident. This was followed by full recovery and any initial effect was considered to be of no toxicological importance.

Food Consumption

At the start of dosing (over Days 5 to 8 of gestation), surviving females from the high dose group showed slightly lower food intake than controls. Taking into view the full subsequent recovery, this finding was deemed not to be of any toxicological relevance.


Water Consumption

Daily visual inspection of water bottles did not identify any overt intergroup differences.

Post Mortem Studies

There were no treatment-related macroscopic findings for any of the females surviving to Day 20 of gestation.

Litter Data and Litter Placental and Fetal Weights

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in fetal growth and development.

Fetal Examination

No treatment-related effects were detected during external examination of the fetuses or in the type and incidence of skeletal or visceral findings.

Conclusion

The oral administration of Bisisobutyryl peroxide (CAS# 3437-84-1) to pregnant rats by gavage during gestation Days 5 to 19, at a dose level of 1000 mg/kg bw/day (first reduced to 600 mg/kg bw/day and then further reduced to 450 mg/kg bw/day) resulted in premature deaths of seven females between Days 6 and 19 of gestation. At 300 mg/kg bw/day, four females were found dead or killedin extremisbetween Days 14 and 19 of gestation. These deaths were considered to be related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. There were no unscheduled deaths amongst females treated with 100 mg/kg bw/day.

The surviving females from the high dose group showed lower body weight gain at the start of dosing, which was associated with slightly lower dietary intake. Recovery was evident thereafter and this initial effect was considered unlikely to be of any toxicological relevance. Excluding these early deaths from the overall assessment, 450 mg/kg bw/day could be established as the ‘No Observed Adverse Effect Level’ (NOAEL) for maternal systemic toxicity and the ‘No Observed Effect Level’ (NOEL) for developmental toxicity within the confines of this study.