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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 11 June 2013 and 02 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Test Guidelines and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Remarks:
No deviations had an adverse impact on the scientific purpose of the study.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dilauroyl peroxide
EC Number:
203-326-3
EC Name:
Dilauroyl peroxide
Cas Number:
105-74-8
Molecular formula:
C24H46O4
IUPAC Name:
dodecanoyl dodecaneperoxoate
Test material form:
solid: flakes
Details on test material:
Identification: Di Lauroyl Peroxide (CAS# 105-74-8)
Description: White Solid Flakes
Chemical Name: Dilauroyl peroxide
Purity: 98.9%
Batch Number: 0905530079
Label: Laurox 0905530079 2KG
Date Received: 19 February 2013
Storage Conditions: Stored in darkness and frozen at -20°C
Expiry Date : 01 June 2019

No correction for purity was made.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
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 220 to 287g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel 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, Harlan UK, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). There were no contaminants that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., 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 computerised system, and print-outs of hourly mean temperatures and humidity were included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20 % respectively (see deviations from Study Plan).

The animals were randomly allocated to treatment groups using a randomisation 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:
corn oil
Details on exposure:
For the purpose of the study the test item was prepared at the appropriate concentrations as a suspension in Corn Oil. The test item was administered daily, from Day 5 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Introduction
The test material concentration in the test samples was determined by gas chromatography (GC) using an external standard technique.

Test item
The test item described in the main part of this study was also used in the analytical standard.

Analytical procedure.
Preparation of standard solutions
Stock solutions of test item in tetrahydrofuran were prepared for external standard calibration. An aliquot, 100 mg of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with tetrahydrofuran 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 tetrahydrofuran with a concentration of 0.1 mg/mL.
On each occasion, standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were diluted with tetrahydrofuran. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with tetrahydrofuran this was then shaken to dissolve. Where necessary, sample solutions were further diluted with tetrahydrofuran to achieve the working concentration.

Instrumental set up
GC system: Agilent Technologies 5890, incorporating autosampler and workstation.
Column: DB-1 (30 m x 00.25 mm id x 0.25 µm film).
Oven temperature program: Oven: 60 °C, for 0 minutes, with 10 °C/minute to 140 °C, then 50 °C/minute to 300 °C, for 5 minutes.
Injection temperature: 250 °C
Flame ionisation detector temperature: 250 °C
Injection volume: 1 µL
Retention time: ~ 8.5 mins

Results
Validation of analytical method
Specificity
The control dose samples and a solvent blank did not significantly affect the chromatogram at the retention time of the test item whose area changed accordingly with known concentration.

Test item formulations
The formulations investigated during the study were found to comprise test item in the range of 92 to 99 % and thus the required content limit of ± 10 % with reference to the nominal content were met.
In addition, the test item was found to be stable in the formulations due to results which met the variation limit of 10 % from the time-zero mean in Harlan Laboratories Ltd Study No. 41206765.
In conclusion, the results indicate the accurate use of the test item in Corm Oil as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
The test item peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of test item and, therefore, the absence of the test item in the vehicle control samples was confirmed.

Discussion
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.
Details on mating procedure:
Not applicable. Time-mated females were used.
Duration of treatment / exposure:
From Day 5 to Day 19 of gestation.
Frequency of treatment:
Daily
Duration of test:
From Day 5 to Day 19 of gestation
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg body weight /day
Basis:
actual ingested
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Observations
Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioural 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. An additional observation was also performed five hours after dosing during the normal working week. 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 animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each 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:
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. 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.

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
Fetal examinations:
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 transferred to 90 % industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope. The remaining fetuses were identified using tags and placed in 70 % IMS in distilled water. The fetuses were eviscerated, processed and the skeletons stained with alizarin red S. The fetuses were examined for skeletal development and anomalies. Following examination fetuses that were examined for skeletal development were placed in 100% glycerol.
Statistics:
All data was summarised in tabular form, including reproductive indices. Where appropriate group mean values was calculated as follows:

Mean 1: Includes data from all surviving pregnant females including those with total resorption
Mean 2: Includes data from surviving pregnant females with live fetuses on Day 20 of gestation

Where appropriate the data was statistically analysed. In-life data was analysed by parametric analysis of variance followed by pairwise comparison where appropriate. Cesarian data was analysed using comparable non-parametric methods.


Pre and Post Implantation Loss
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 fetuses)/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

Statistical Analysis
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Female body weight change, food consumption and gravid uterus weight: 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 cesarean 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<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)



Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
There were no treatment-related deaths.

One female treated with 100 mg/kg bw/day was killed in extremis on Day 9. Macroscopic observations revealed evidence of a physical trauma following the dosing procedure therefore this death was considered unrelated to test item toxicity.


Clinical Observations
Females treated with 1000 and 300 mg/kg bw/day showed isolated incidences of increased salivation on either Day 18 or Day 19. No such effects were detected in females treated with 100 mg/kg bw/day. Observations of this nature are commonly observed following the oral administration of an unpalatable test item formulation and in isolation are considered not to be of toxicological significance.

The female that was killed in extremis on Day 9 showed red/brown staining around the snout, hunched posture, lethargy, pilo-erection, decreased respiratory rate and a mass near the front left leg.


Body Weight
No adverse effect on body weight development was detected.

Females treated with 300 mg/kg bw/day showed a statistically significant reduction in body weight gain and cumulative body weight gain between Days 5 and 6. In the absence of a true dose related response the intergroup difference was considered of no toxicological importance.

Females treated with 100 mg/kg bw/day showed a statistically significant increase in body weight gain between Days 7 and 8. An increase in body weight gain is not considered to represent an adverse effect of treatment.


Food Consumption
No adverse effect on dietary intake was detected. Statistical analysis of the data did not reveal any significant differences.


Water Consumption
Daily visual inspection of water bottles did not reveal any overt intergroup differences.


Post Mortem Studies
No treatment-related macroscopic abnormalities were detected in treated females at terminal kill.

The female that was killed in extremis had stained eyes and snout, yellow fluid beneath the left forelimb and a hole in the esophagus.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Responses
Litter Data and Litter Placental and Fetal Weights
There was no treatment related effects on in-utero offspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and post-implantation losses. There were also no adverse effects on pre-implantation losses or in sex ratio.

Litters from females treated with 1000 mg/kg bw/day showed a statistically significant increase in the number of late resorptions. This was considered to be due to one litter which showed an increased number of late resorptions. In isolation and in the absence of any associated effect on total deaths or live litter size the intergroup difference was considered not to be of toxicological significance.


Fetal Examination
For all dose groups, there were no significant treatment-related trends in the proportion of fetuses (or litters) with evidence of visceral or skeletal anomalies. The type of external, visceral and skeletal anomalies, were those commonly observed for this type of study. There were no findings that were considered to represent any known malformations.

Statistical analysis of the data did not reveal any significant differences.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
other: developmental toxicity
Remarks:
No toxicological significant changes were detected in the offspring parameters measured.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Summary of Female Performance

Category

Number of Females at Dose Level (mg/kg bw/day)

0 (Control)

100

300

1000

Initial Group Size

24

24

24

24

Pregnant

24

24

24

24

Killedin extremis

0

1

0

0

 

Summary Incidence of Daily Clinical Observations

Dose Level (mg/kg bw/day)

Number of Animals

Clinical Observations

Number Showing Effect
(Dayspost coitumaffected)

0 (Control)

24

Generalised fur loss

2 (17-20)

100

24/23

Red/brown stained snout

Red/brown staining around eyes

Hunched posture

Lethargy

Pilo-erection

Decreased respiratory rate

Mass near front left leg

Killedin extremis

1 (9)

300

24

Increased salivation

3 (18-19)

1000

24

Increased salivation

2 (19)

 

Group Mean Body Weight Values

Dose Level (mg/kg bw/day)

Number of Animals

 

Body Weight (g) at Day of Gestation

3

5

6

7

8

11

14

17

20

0 (Control)

24

mean

251.9

264.6

268.3

273.9

277.6

296.3

311.9

343.0

387.5

sd

17.2

20.0

19.6

20.3

20.3

22.6

23.7

28.3

30.2

100

24/23#

mean

248.8

260.3

263.5

268.7

275.3

291.6

310.0

343.0

389.5

sd

12.6

13.1

13.3

13.8

15.4

14.2

16.7

19.6

21.5

300

24

mean

247.4

260.1

261.5

267.2

272.5

288.5

307.7

338.8

384.6

sd

11.9

12.1

12.7

13.3

13.7

15.8

16.1

17.8

20.7

1000

24

mean

249.5

261.4

265.2

271.6

275.3

294.4

311.6

343.1

387.8

sd

12.6

12.5

12.4

13.1

14.5

15.8

17.9

19.9

22.3

 

Group Mean Gravid Uterus Weight and Adjusted Body Weight and Body Weight Change Values

Dose Level (mg/kg bw/day)

Number
of
Animals

 

Body Weight (g) on Days of Gestation

Body Weight Change (g) during Days of Gestation

Gravid Uterus Weight
(g)

Adjusted
Body Weight (g)
 Day 20

Adjusted
Body Weight (g)
Change
Days 5-20

5

20

5-20

0 (Control)

24

mean

264.6

387.5

123.0

82.618

304.9

40.3

sd

20.0

30.2

18.5

16.135

21.7

10.2

100

23#

mean

260.3

389.5

128.5

82.790

306.7

45.7

sd

13.1

21.5

13.7

14.853

17.6

11.1

300

24

mean

260.1

384.6

124.5

78.503

306.1

46.0

sd

12.1

20.7

14.4

10.799

21.1

15.2

1000

24

mean

261.4

387.8

126.4

78.678

309.1

47.7

sd

12.5

22.3

14.1

12.857

19.7

12.2

 

Group Mean Food Consumption Values

Dose Level (mg/kg bw/day)

Number
of
Animals

 

Food Consumption (g/rat/day) between Days of Gestation

3 - 5

5 - 8

8 - 11

11 - 14

14 - 17

17 - 20

0 (Control)

24

mean

24.9

22.1

24.3

24.4

25.4

24.6

sd

2.6

2.1

2.9

3.0

2.6

2.2

100

24/23#

mean

24.1

21.2

22.6

23.4

25.8

25.0

sd

2.1

1.7

2.3

2.5

2.5

2.3

300

24

mean

24.6

20.9

22.8

24.0

25.6

24.0

sd

2.6

2.0

2.1

2.1

2.4

2.1

1000

24

mean

25.1

21.2

23.2

23.9

26.0

23.8

sd

2.7

2.6

2.6

2.3

2.5

2.5

#n = 23 from Day 9 onwards

Applicant's summary and conclusion

Conclusions:
The oral administration of Di Lauroyl Peroxide (CAS# 105-74-8) to pregnant rats by oral gavage during organogenesis at dose levels of 100, 300 and 1000 mg/kg/day did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg bw/day.

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.
Executive summary:

Introduction

The study was performed according to the study plan presented in Appendix13and 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: 

·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

·        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 by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Corn oil) to serve as a control.

 

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

 

All 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 preserved in Industrial Methylated Spirit (IMS) and then following examination for skeletal development transferred into 100% glycerol. The remaining half were preserved in Bouin’s solution and transferred to 90% IMS in distilled water and examined viscerally.

 

Results…….

Mortality

There were no treatment related deaths.

 

Clinical Observations

Females treated with 1000 and 300 mg/kg bw/day showed isolated incidences of increased salivation on either Day 18 or Day 19. No such effects were detected in females treated with 100 mg/kg bw/day.

 

Body Weight

No treatment related effects in body weight development were detected.

 

Food Consumption

No treatment related effects were detected in food consumption.

 

Water Consumption

No adverse effect on water consumption was detected.

 

Post Mortem Studies

No treatment related macroscopic abnormalities were detected.

 

Litter Data and Litter Placental and Fetal Weights

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

 

Fetal Examination

No treatment-related effects were detected on skeletal development or in the type and incidence of skeletal or visceral findings.

 

Conclusion

The oral administration of Di Lauroyl Peroxide (CAS# 105-74-8) to pregnant rats by oral gavage during organogenesis at dose levels of 100, 300 and 1000 mg/kg/day did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg bw/day.

 

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg/day.