Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 11 January 2017 and 03 May 2017
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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear, colourless liquid
Details on test material:
Identity: 1,1,3,3-tetramethylbutyl hydroperoxide
CAS No.: CAS 5809-08-5
Batch-No.: 1010519063
Molecular Weight: 146.2 g/mol
Purity: 91.1 % (dose calculation adjusted to purity)
Stability in Solvent: Not indicated by the sponsor
Storage Conditions: At +2 to +8 °C
Expiration Date: November 01, 2012
Specific details on test material used for the study:
Description: Clear colorless liquid
Chemical Name: 1,1,3,3-tetramethylbutyl hydroperoxide
CAS Number : 5809-08-5
EC Name: 1,1,3,3-tetramethylbutyl hydroperoxide
Purity: 92.5%
Batch Number: 1504510829
Label: Trigonox TMBH-L -5ºC MIN, 25ºC MAX
Date Received: 21 June 2016
Storage Conditions: Approximately 4ºC in the dark
Expiry Date: 06 January 2018

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals 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 189 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. Certificates of analysis of the batches of diet used are given in Annex 2. 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:
PREPARATION OF DOSING SOLUTIONS:

For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. Formulations were prepared in two batches each of 14 days and made in advance of the first day of use and stored at approximately 4ºC in the dark.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services (Harlan Study Number: 41104766 and Envigo Study Number: CN35PW). Results showed the formulations to be stable for at least nineteen days.
Samples were taken of each test item formulation and were analyzed for concentration of 1,1,3,3-tetramethylbutyl hydroperoxide (CAS 5809-08-5) at Envigo Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 94-101% of the nominal concentration, confirming accurate formulation.

The homogeneity was detennined with respect to the concentration of 1.1,3,3-tetramethylbutyl bydroperoxide (CAS#5809-08-5) (test item) in Arachis oil formulations at a nominal concentration of 150 mg/mL.
The concentrations of Test Item in Arachis oil formulations analyzed during the study.
The stability and validatioo was determined under Harlan Srudy Nwuber 4 1104766 at concentrations of 3.75 and 250 mg/mL. The test item showed to be stable for up to 19 days when stored at approltimately 4 °C in the dark.
The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision during Harlan study 41104766. The homogeneity was confirmed for Test Item in Arachis oil formulations at a nominal concentration of 150 mg/mL. The mean concentrations of Test Item in test formulations analyzed for the study were within ± 10%, of nominal concentrations confirming accurate formulation.
Details on mating procedure:
Females were time-mated with sexually proven males of the same strain by the animal supplier. The day of observation of positive evidence of mating will be designated Day 0 of gestation. The animal supplier will identify mated females and provide identification details of their respective mating partners.
Duration of treatment / exposure:
The test item was administered from Day 5 to Day 19 of gestation.
Frequency of treatment:
Daily.
Duration of test:
20 days from Day 0 of gestation to Day 20.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen in collaboration with the Sponsor and are based on previous toxicity data including a 28 day oral (gavage) toxicity study in the rat (Harlan Study Number: 41104766) and a 90 day oral (gavage) toxicity study in the rat (Envigo Study Number: CN35PW). 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 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.

Necropsy
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, the stomachs were retained and any macroscopic abnormalities were recorded.
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

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
Fetal examinations:
The fetuses were killed by subcutaneous injection of a suitable barbiturate agent. 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 into 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:
Body weights, 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<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:
((number of corpora lutea - number of implantations) / mumber 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 males fetuses / total number of fetuses) x 100
 

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were considered to be no clinical signs apparent that were specifically related to systemic toxicity of the test item.
Two animals treated with 600 mg/kg bw/day exhibited sporadic instances of hunched posture during the treatment period which became apparent from Day 6 of gestation.
Increased salivation was noted in all animals treated with 600 mg/kg bw/day from Days 6 to 19, in seventeen animals treated with 200 mg/kg bw/day from Days 8 to 19 and in one animal treated with 50 mg/kg bw/day.
Noisy respiration was noted in three animals treated with 600 mg/kg bw/day on three separate occasions (persisting for one day only) Days 10, 14 and 18.
Generalized fur loss was noted in one animal treated with 50 mg/kg bw/day from Days 13 to 20.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females treated with 600 mg/kg bw/day showed a statistically significant reduction
(p<0.001 - p<0.01) in body weight gain between Days 5 and 6 of gestation, with actual body weight losses being evident in the majority of females. Signs of recovery were noted from Days 7 to 14 and even a statistically significant increase (p<0.01) in body weight was noted in these animals from Days 7 to 8, however, reductions in body weight gain were again apparent between Days 14 and 20 (which may be a reflection of lower litter weights/ contribution of the gravid uterus) which achieved statistical significance (p<0.01-p<0.001). Body weight and body weight gain when adjusted for gravid uterus weight was also statistically significantly reduced (p<0.001) (-11 and -55% respectively) in these females.
Females treated with 200 mg/kg bw/day exhibited slight reductions in body weight gains between Days 5 and 8 (which did not achieve statistical significance), however, this did result in a statistically significant reduction in cumulative body weight gain from Days 5 to 7 and 5 to 8. Recovery was evident thereafter and by Day 20, overall body weight gain was comparable to controls. A statistically significant (p<0.05) reduction in body weight gain
(-17%) after the adjustment for the contribution of the gravid uterus was still noted at the end of the treatment period.
Body weight gain during gestation, including after adjustment for the contribution of the gravid uterus, was considered to be unaffected by treatment at 50 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reductions (p<0.05 - p<0.001) in food consumption were evident in females treated with 600 mg/kg bw/day throughout the treatment period.
A slight reduction in food consumption was noted in females treated with 200 mg/kg bw/day from Days 5 to 8 which achieved statistical significance (p<0.01). However, recovery was evident thereafter such that food consumption was comparable to control.
No such effects were evident in females treated with 50 mg/kg bw/day.
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:
effects observed, treatment-related
Description (incidence and severity):
All females treated with 600 mg/kg bw/day exhibited thickening and/or sloughing of the non-glandular region of the stomach. Isolated occurrences of yellow colored contents in the stomach and raised white patches on the non-glandular region of the stomach were also apparent.
No macroscopic abnormalities considered to be related to treatment with the test item were noted in any other animal.
One female treated with 50 mg/kg bw/day exhibited generalized fur loss.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
not specified
Description (incidence and severity):
Not applicable in rats
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See table 'Group Mean Litter Data Values' in section 'any other information on results'
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See table 'Group Mean Litter Data Values' in section 'any other information on results'
Early or late resorptions:
no effects observed
Description (incidence and severity):
See table 'Group Mean Litter Data Values' in section 'any other information on results'
Dead fetuses:
no effects observed
Description (incidence and severity):
See table 'Group Mean Litter Data Values' in section 'any other information on results'
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Females killed on Day 20 of gestation
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Females killed on Day 20 of gestation
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
See table 'Group Mean Litter Data Values' in section 'any other information on results'
Other effects:
no effects observed
Details on maternal toxic effects:
There was no effect of maternal exposure to 50, 200 or 600 mg/kg bw/day of the test item on litter data, as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and post implantation losses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
gross pathology
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
early or late resorptions
dead fetuses
changes in number of pregnant

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg bw/day, mean fetal weight for both sexes was statistically significantly lower (p<0.05 - p<0.001) than control (-9.8 % male and -11.4 % female), resulting in statistically significantly lower (p<0.05) mean litter weights (-12.4 %). Placental weights were also statistically significantly reduced (p<0.01) in these females (-10.2 %).
No adverse effects on fetal, litter or placental weights were detected in females treated with 50 or 200 mg/kg bw/day.
At 200 mg/kg bw/day, mean male and female fetal weights were slightly lower than control
(-2.7 % and -5.3 % respectively), however, only females achieved statistical significance (p<0.05), total litter weights and placental weights actually exceeded control.

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:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
Description (incidence and severity):
Animals terminated on Day 20 of gestation.
External malformations:
effects observed, treatment-related
Description (incidence and severity):
An increased incidence of small fetuses was evident from females treated with 600 mg/kg bw/day which resulted in a statistically significant increase in total number of fetuses with external findings at necropsy.
With the exception of one small fetus from a female treated with 200 mg/kg bw/day, no such findings were evident in litters from females treated with 200 or 50 mg/kg bw/day.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg bw/day, there was clear effect of treatment on a large number of ossification parameters affecting most regions of the skeleton, with the number of affected fetuses/litters increased compared with control and differences frequently attaining statistical significance (p<0.01).
These parameters included unossified areas of the frontal and incomplete ossification of the occipital (Supra Occipital) bones of the skull, incomplete ossification of the cervical (neural) arch and thoracic centrum, incomplete ossification of the sacral (neural) arch and no ossification of the metacarpals. There was also a lower incidence of fetuses showing ossification of the ventral arch of vertebra 1, ossification of one or more fore paw phalanges and metatarsal 1st ossified.
At 200 mg/kg bw/day, an increased incidence of fetuses/litters showing incomplete ossification of the thoracic centrum was evident.
At 50 mg/kg bw/day, an increased incidence of fetuses/litters showing incomplete ossification of the cervical (neural) arch was evident.
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 50, 200 or 600 mg/kg bw/day.
Other effects:
no effects observed

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
other: fetal and placental weights
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: skeletal: generalised pattern of delays in ossification

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
not specified

Any other information on results incl. tables

Key

M

Male

F

Female

sd

Standard deviation

n

Number of animals/litters

NF

Number of fetuses

NL

Number of litters

%†

Group mean percent

N/A

Not applicable

NP

Not pregnant

V

Viable fetuses+

FWT

Fetal weight

PWT

Placental weight

C

Cervix

ED

Early death

LD

Late death

DF

Dead Fetus

-

No data available

 

 

Statistical Footnotes:

*

Significantly different from control group p<0.05

**

Significantly different from control group p<0.01

***

Significantly different from control group p<0.001


+= viable fetuses are identified by assigned fetal number

Summary of Female Performance

Category

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

0 (Control)

50

200

600

Initial Group Size

24

24

24

24

Pregnant

24

24

22

24

Non-Pregnant

0

0

2

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

No abnormalities detected

 

50

24

Increased salivation

Generalized fur loss

1 (14)

1 (13-20)

200

24

Increased salivation

17 (8-19)

600

24

Increased Salivation

Hunched posture

Noisy respiration

24 (6-19)

2 (6-14, 16-19)

3 (10, 14, 18)

 


 

Group Mean Body Weight Values

Dose Level (mg/kg bw/day)

 

Body Weight (g) on Day of Gestation

3

5

6

7

8

11

14

17

20

0 (Control)

mean

244.6

259.6

262.5

269.2

274.0

293.5

313.7

345.9

391.6

sd

22.4

22.8

23.3

23.5

23.7

24.7

25.2

27.5

29.5

n

24

24

24

24

24

24

24

24

24

50

mean

246.1

262.0

265.0

271.4

277.0

294.9

315.6

347.8

391.9

sd

19.6

21.3

21.2

19.9

20.5

21.0

21.8

23.6

27.8

n

24

24

24

24

24

24

24

24

24

200

mean

243.1

258.7

260.1

265.1

269.4

288.2

308.1

340.6

387.2

sd

16.6

18.9

18.9

18.6

18.2

21.1

24.2

27.1

33.4

n

22

22

22

22

22

22

22

22

22

600

mean

242.9

257.2

251.0

248.3**

255.8*

272.5**

293.5*

317.9**

353.6***

sd

20.1

22.3

21.9

22.2

24.0

23.8

24.1

28.9

32.6

n

24

24

24

24

24

24

24

24

24

 


 

Group Mean Body Weight Change Values

Dose Level (mg/kg bw/day)

 

Body Weight Change (g) during Days of Gestation

3 to 5

5 to 6

6 to 7

7 to 8

8 to 11

11 to 14

14 to 17

17 to 20

0 (Control)

mean

15.0

2.9

6.7

4.8

19.5

20.2

32.2

45.8

sd

3.7

2.9

4.3

3.3

5.6

3.4

5.5

5.5

n

24

24

24

24

24

24

24

24

50

mean

15.9

3.0

6.4

5.7

17.8

20.8

32.1

44.2

sd

4.8

3.7

3.5

3.6

4.5

4.3

4.9

7.0

n

24

24

24

24

24

24

24

24

200

mean

15.6

1.4

5.0

4.3

18.9

19.9

32.5

46.6

sd

3.2

4.3

5.4

3.7

5.5

5.4

4.6

8.1

n

22

22

22

22

22

22

22

22

60

mean

14.3

-6.2***

-2.8***

7.5**

16.8

21.0

24.4**

35.7***

sd

6.4

5.3

4.9

8.0

5.1

6.0

8.8

9.9

n

24

24

24

24

24

24

24

24

 

Dose Level (mg/kg bw/day)

 

Cumulative Body Weight Change (g) from Day 5 of Gestation

6

7

8

11

14

17

20

0 (Control)

mean

2.9

9.6

14.4

33.9

54.1

86.3

132.0

sd

2.9

4.6

4.2

7.0

7.0

10.4

13.9

n

24

24

24

24

24

24

24

50

mean

3.0

9.3

15.0

32.8

53.6

85.7

129.9

sd

3.7

4.0

5.5

7.3

8.9

10.1

14.8

n

24

24

24

24

24

24

24

200

mean

1.4

6.4*

10.6**

29.5

49.4

81.9

128.5

sd

4.3

3.8

4.1

5.0

7.7

10.7

16.8

n

22

22

22

22

22

22

22

600

mean

-6.2***

-8.9***

-1.4***

15.4***

36.4***

60.8***

96.5***

sd

5.3

5.0

8.2

8.8

10.0

14.6

18.6

n

24

24

24

24

24

24

24


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

Dose Level (mg/kg bw/day)

 

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 Change (g)
5-20

5

20

5-20

0 (Control)

mean

259.6

391.6

132.0

83.128

308.5

48.9

sd

22.8

29.5

13.9

13.289

25.1

11.3

n

24

24

24

24

24

24

50

mean

262.0

391.9

129.9

84.342

307.6

45.5

sd

21.3

27.8

14.8

12.882

20.3

10.7

n

24

24

24

24

24

24

200

mean

258.7

387.2

128.5

88.093

299.1

40.4*

sd

18.9

33.4

16.8

9.669

26.5

12.8

n

22

22

22

22

22

22

600

mean

257.2

353.6

96.5

73.982*

279.2***

21.9***

sd

22.3

32.6

18.6

13.211

24.9

13.1

n

24

24

24

23

23

23

 


 Group Mean Food Consumption Values

Dose Level (mg/kg bw/day)

 

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

3 - 5

5 - 8

8 - 11

11 - 14

14 - 17

17 - 20

0 (Control)

mean

24.9

22.8

24.5

25.1

27.1

26.7

sd

2.5

2.5

2.5

2.2

2.8

2.3

n

24

24

24

24

24

24

50

mean

25.5

23.2

24.5

25.7

27.3

26.5

sd

2.2

1.5

1.7

2.3

2.5

2.1

n

24

24

24

24

24

24

200

mean

24.6

20.6**

23.7

24.2

27.1

25.9

sd

2.2

1.9

2.9

2.9

3.3

3.3

n

22

22

22

22

22

22

600

mean

24.0

12.2***

20.1***

22.8*

23.3***

24.6*

sd

3.6

2.6

2.3

3.5

4.0

3.0

n

24

24

24

24

24

24

 


 Summary Incidence of Necropsy Findings

 

Dose Level (mg/kg bw/day)

0 (Control)

50

200

600

INTERIM DEATH

 

 

 

 

 

 

 

 

 

Number of animals examined

24

24

24

24

Generalized fur loss

0

1

0

0

Stomach – non-glandular region – thickened

0

0

0

23

Stomach – non-glandular region – sloughing

0

0

0

24

Stomach – yellow colored contents

0

0

0

3

Stomach – raised white patches

0

0

0

1

No abnormalities detected

24

23

24

0

 

Group Mean Litter DataValues

Dose Level (mg/kg bw/day)

 

Number of Corpora Lutea

Number of Implants

Number of Embryonic/Fetal Deaths

Implantation Loss
%

Number of Live Implants

%
Male
Fetuses

Mean Male Fetal Weight (g)

Mean Female Fetal Weight (g)

Mean Fetal Weight (g)

MeanPlacentalWeight
(g)

Litter Weight (g)

TotalPlacentalWeight
(g)

Early

Late

Total

Pre

Post

Male

Female

Total

0 (Control)

mean

14.5

13.4

0.2

0.2

0.4

7.1

3.2

6.3

6.7

13.0

48.8

4.264

4.069

4.161

0.588

53.742

7.585

sd

2.3

2.1

0.5

0.7

1.0

10.1

8.8

1.7

2.1

2.5

11.4

0.353

0.328

0.325

0.072

9.761

1.477

n

23

23

24

24

24

23

23

24

24

24

24

24

24

24

24

24

24

50

mean

14.4

13.5

0.2

0.2

0.4

6.5

3.1

6.5

6.5

13.0

50.5

4.290

4.081

4.185

0.567

54.483

7.392

sd

1.3

2.0

0.5

0.4

0.7

11.2

5.1

2.4

2.5

2.1

17.8

0.268

0.248

0.255

0.055

9.146

1.359

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

200

mean

15.3

14.5

0.2

0.1

0.4

4.8

2.5

7.3

6.9

14.2

51.6

4.147

3.854*

4.003

0.562

56.660

7.962

sd

1.6

1.7

0.5

0.4

0.6

7.5

4.3

1.6

2.0

1.7

11.1

0.288

0.314

0.291

0.076

7.167

1.442

n

22

22

22

22

22

22

22

22

22

22

22

22

22

22

22

22

22

600

mean

14.8

13.4

0.2

0.5

0.7

9.5

5.2

6.2

6.5

12.7

48.2

3.845*

3.606*

3.723***

0.528**

47.100*

6.684

sd

2.0

2.2

0.4

0.8

0.9

10.8

7.1

2.0

2.1

2.3

13.9

0.305

0.294

0.299

0.070

8.476

1.410

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

 


 Summary Incidence of Fetal External Findings

External Findings

Dose level (mg/kg bw/day)

0 (Control)

50

200

600

Number of fetuses (litters) examined

312 (24)

313 (24)

312 (22)

305 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Total Number Affected

1

1

0.3

0

0

0.0

1

1

0.4

19

6

5.8*

Small

1

1

0.3

0

0

0.0

1

1

0.4

18

5

5.5

Hematoma - lower left side of back

0

0

0.3

0

0

0.0

0

0

0.0

1

1

0.3

 

 

Summary Incidence of Fetal Visceral Findings

Visceral Findings

Dose Level (mg/kg bw/day)

0 (Control)

50

200

600

Number of Fetuses (litters) Examined

162 (24)

161 (24)

160 (22)

158 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

EXTERNAL/GENERAL

 

 

 

 

 

 

 

 

 

 

 

 

Hemorrhage – kidney cortex - R

1

1

0.7

0

0

0.0

0

0

0.0

0

0

0.0

HEAD

 

 

 

 

 

 

 

 

 

 

 

 

Tongue - short

1

1

0.6

0

0

0.0

0

0

0.0

1

1

0.6

Rugae - non-uniform patterning

17

10

10.7

7

6

5.2

8

8

5.5

10

6

6.4

ABDOMEN

 

 

 

 

 

 

 

 

 

 

 

 

Liver - additional lobe between right and left median

0

0

0.0

1

1

0.6

0

0

0.0

2

2

1.1

Umbilical artery - left-sided

0

0

0.0

1

1

0.6

1

1

0.6

2

2

1.2

Urinary bladder - distended with fluid or gas

0

0

0.0

0

0

0.0

2

1

1.1

2

1

1.2

Testis - partially undescended

1

1

0.5

3

2

1.6

0

0

0.0

0

0

0.0

Ureter - kinked

18

14

10.9

9

8

5.3

20

10

12.1

13

10

8.5

Ureter - dilated - Slight/Severe

5

5

3.3

5

5

2.9

11

8

6.8

8

6

5.0

Renal pelvic cavitation - increased - Slight/Severe

10

7

5.9

5

4

3.0

13

8

7.7

4

3

2.4

Renal papilla - absent

3

2

2.0

2

2

1.1

1

1

0.6

2

2

1.5

THORAX

 

 

 

 

 

 

 

 

 

 

 

 

Thymus - lobe partially undescended

2

2

1.6

5

5

2.9

2

2

1.4

15

8

9.9

Total

37

20

22.6

25

17

15.7

36

18

22.5

44

19

27.9


 Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

50

200

600

Number of Fetuses (litters) Examined

150 (24)

152 (24)

152 (22)

147 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

SKULL

 

 

 

 

 

 

 

 

 

 

 

 

Fontanelle (anterior) - large

1

1

0.8

0

0

0.0

2

1

1.5

3

3

1.9

Nasal - incomplete ossification

10

6

6.7

16

9

10.6

15

7

9.6

26

10

17.4

Nasal/Frontal - sutural bone

0

0

0.0

4

1

2.4

1

1

0.8

1

1

0.6

Frontal - incomplete ossification

2

2

1.4

4

3

2.6

2

2

1.3

6

2

3.8

Frontal - unossified area

1

1

0.5

1

1

0.7

4

3

2.5

12

8

7.8**

Parietal - incomplete ossification

6

4

4.1

10

5

7.0

8

6

5.4

11

8

7.1

Parietal - unossified area(s)

0

0

0.0

1

1

0.6

0

0

0.0

0

0

0.0

Interparietal - incomplete ossification

18

11

11.4

26

11

17.1

27

12

17.7

42

16

29.8

Interparietal - unossified area(s)

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.7

Occipital (Supra-occipital) - incomplete ossification

8

4

5.3

20

9

13.3

11

9

7.3

44

14

29.2**

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

6

5

3.8

3

3

2.0

3

2

2.2

10

7

6.2

Exoccipital - fused to 1stcervical (neural) arch

0

0

0.0

0

0

0.0

1

1

0.8

0

0

0.0

Squamosal - incomplete ossification

5

4

3.4

17

8

11.9

16

9

10.1

9

5

5.7

Squamosal - unossified area(s)

3

2

1.9

2

2

1.4

7

4

4.5

2

2

2.8

Jugal - incomplete ossification

2

2

1.2

4

4

2.8

1

1

0.6

1

1

0.6

Jugal - misshapen

0

0

0.0

0

0

0.0

1

1

0.8

0

0

0.0

Zygomatic process of maxilla - incomplete ossification

7

4

4.6

12

6

8.4

17

9

11.7

3

3

2.0

Zygomatic process of squamosal - incomplete ossification

0

0

0.0

1

1

0.7

1

1

0.8

1

1

0.7

Zygomatic process of squamosal - fused to jugal

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6


 

Summary Incidence of Fetal Skeletal Findings (continued)

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

50

200

600

Number of Fetuses (litters) Examined

150 (24)

152 (24)

152 (22)

147 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

SKULL (continued)

 

 

 

 

 

 

 

 

 

 

 

 

Premaxilla - incomplete ossification

0

0

0.0

0

0

0.0

2

1

1.1

1

1

0.5

Hyoid - incomplete ossification

6

5

4.0

14

11

9.3

7

5

4.5

7

6

4.7

Hyoid - not ossified

5

4

2.8

7

6

4.7

11

9

7.0

12

9

8.1

Mandible - incomplete ossification

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Tympanic annulus - incomplete ossification

0

0

0.0

0

0

0.0

1

1

0.8

1

1

0.6

Presphenoid - incomplete ossification

1

1

0.6

1

1

0.6

0

0

0.0

0

0

0.0

Basisphenoid - incomplete ossification

2

2

1.5

0

0

0.0

2

2

1.3

0

0

0.0

VERTEBRAL COLUMN

 

 

 

 

 

 

 

 

 

 

 

 

Odontoid - ossification present

1

1

0.7

0

0

0.0

0

0

0.0

0

0

0.0

Ventral arch of vertebra 1 - ossification present

54

20

36.0

41

18

27.9

60

22

39.3

22

13

14.4**

Cervical (neural) arch - incomplete ossification

0

0

0.0

6

4

3.9*

4

3

2.8

12

8

7.4**

Thoracic centrum - incomplete ossification

0

0

0.0

2

2

1.3

4

4

2.5*

9

8

5.6**

Thoracic centrum - not ossified

0

0

0.0

0

0

0.0

1

1

0.8

1

1

0.6

Thoracic centrum - bipartite ossification

6

5

3.7

1

1

0.6

3

3

1.9

5

4

3.0

Thoracic centrum - dumb-bell-shaped

17

13

11.8

10

7

7.1

23

14

14.6

29

14

18.8

Thoracic centrum - asymmetrically ossified

6

4

3.5

0

0

0.0

2

2

1.1

1

1

0.5

Thoracic (neural) arch - incomplete ossification

0

0

0.0

1

1

0.7

0

0

0.0

0

0

0.0

Lumbar centrum - incomplete ossification

0

0

0.0

0

0

0.0

2

2

1.4

1

1

0.6

Lumbar centrum - bipartite ossification

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Lumbar centrum - dumb-bell-shaped

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

 

Summary Incidence of Fetal Skeletal Findings (continued)

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

50

200

600

Number of Fetuses (litters) Examined

150 (24)

152 (24)

152 (22)

147 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

VERTEBRAL COLUMN (continued)

 

 

 

 

 

 

 

 

 

 

 

 

Sacral (neural) arch - incomplete ossification

12

5

7.9

12

6

8.2

8

5

4.8

15

8

10.6

Caudal vertebrae - less than 4 ossified

19

11

11.5

21

10

14.3

16

8

10.3

35

14

23.1

RIBS

 

 

 

 

 

 

 

 

 

 

 

 

Ossification centre - associated with 7th cervical vertebra

0

0

0.0

1

1

0.6

1

1

0.6

1

1

0.6

Ossification centre - associated with 1st lumbar vertebra

8

5

5.3

2

2

1.4

7

6

4.4

13

7

7.8

One or more ribs - wavy

1

1

0.7

0

0

0.0

0

0

0.0

0

0

0.0

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

0

0

0.0

1

1

0.6

0

0

0.0

Rib - rudimentary

2

1

2.8

1

1

0.8

0

0

0.0

0

0

0.0

Rib - fused

0

0

0.0

1

1

0.6

0

0

0.0

0

0

0.0

Rib - no articulation point

0

0

0.0

0

0

0.0

1

1

0.8

1

1

0.6

Costal cartilage - misaligned

4

4

2.5

5

5

3.1

2

2

1.4

5

5

3.0

Costal cartilage - not fused to sternebra

10

5

8.6

18

10

12.7

15

10

9.8

17

12

13.1

STERNEBRAE

 

 

 

 

 

 

 

 

 

 

 

 

Sternebra - incomplete ossification

0

0

0.0

0

0

0.0

2

2

1.3

4

4

2.6

Sternebra - not ossified

1

1

0.5

1

1

0.6

1

1

0.8

2

2

1.3

Sternebra - bipartite ossification

2

2

1.0

0

0

0.0

0

0

0.0

2

2

1.3

Sternebra - misaligned

8

7

4.7

2

2

1.3

3

3

2.1

8

8

5.2

Sternebra - fused

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Xiphoid cartilage - partially split

12

9

8.0

9

7

5.8

11

5

7.7

10

7

7.1


 

Summary Incidence of Fetal Skeletal Findings (continued)

Skeletal Findings

Dose Level (mg/kg bw/day)

0 (Control)

50

200

600

Number of Fetuses (litters) Examined

150 (24)

152 (24)

152 (22)

147 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

PECTORAL GIRDLE

 

 

 

 

 

 

 

 

 

 

 

 

Scapula - misshapen (comment on region)

5

4

3.6

3

3

1.9

4

4

2.5

7

5

4.1

PELVIC GIRDLE

 

 

 

 

 

 

 

 

 

 

 

 

Ischium - incomplete ossification

0

0

0.0

1

1

0.7

0

0

0.0

0

0

0.0

Pubis - not ossified

0

0

0.0

0

0

0.0

2

1

1.3

1

1

0.6

Pubis - incomplete ossification

3

3

1.6

5

3

3.1

5

5

3.4

3

2

1.9

FORELIMBS

 

 

 

 

 

 

 

 

 

 

 

 

Metacarpal - not ossified

25

12

16.2

23

8

16.4

26

12

16.4

64

21

43.2**

Metacarpal - incomplete ossification

0

0

0.0

2

2

1.3

0

0

0.0

1

1

0.7

Forepaw phalanges - 1 or more - ossified

42

15

30.3

45

13

29.7

28

14

18.8

9

6

5.7**

Humerus - incomplete ossification

1

1

0.6

2

2

1.4

0

0

0.0

3

2

1.9

HINDLIMBS

 

 

 

 

 

 

 

 

 

 

 

 

Metatarsal - 1st - ossified

16

7

10.4

10

7

6.9

7

3

4.5

0

0

0.0**

Metatarsal - not ossified

0

0

0.0

0

0

0.0

1

1

0.8

0

0

0.0

Metatarsal - incomplete ossification

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Femur - incomplete ossification

10

7

6.4

14

8

9.3

11

6

7.1

12

9

8.4

Total

130

24

86.8

132

24

87.9

132

22

86.8

132

24

90.0

 



NOTE: a fetus may appear in more than one category

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of 1,1,3,3-tetramethylbutyl hydroperoxide (CAS#5809-08-5) to pregnant rats from gestation Days 5 to 19, at dose levels of 50, 200 or 600 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an effect on food consumption at 600 mg/kg bw/day. Slight effects on body weight development were noted at 200 mg/kg bw/day, however, these were considered not to be toxicologically significant. No similar effects were apparent at 50 mg/kg bw/day. Consequently, 200 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female and 50 mg/kg bw/day was considered to represent the No Observed Effect Level (NOEL) for the pregnant female.
In-utero survival of the developing conceptus was unaffected by maternal treatment with 600 mg/kg bw/day, although reduced fetal and placental weights and an increased incidence of skeletal findings indicated an adverse effect on fetal growth. No changes in the measured fetal parameters or embryofetal development were detected at 200 or 50 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 200 mg/kg bw/day.
Executive summary:

Introduction

The study was performed 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 50, 200, and 600 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil BP) 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 weights, 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

There were no unscheduled deaths during the study.

Clinical Observations

There were considered to be no clinical signs apparent that were specifically related to systemic toxicity of the test item.

Body Weight

Females treated with 600 mg/kg bw/day showed a statistically significant reduction in body weight gain between Days 5 and 7 of gestation, with actual body weight losses being evident in the majority of females on these days. Signs of recovery were noted from Days 7 to 14, however, reductions in body weight gain were again apparent between Days 14 and 20 which achieved statistical significance. Body weight and body weight gain when adjusted for gravid uterus weight were also statistically significantly reduced (-11 and -55% respectively) in these females.

No toxicologically significant effects were detected in females treated with 50 and 200 mg/kg bw/day.

Food Consumption

Reductions in food consumption were evident in females treated with 600 mg/kg bw/day throughout the treatment period. No adverse effects of treatment were apparent in females treated with 200 or 50 mg/kg bw/day.

Water Consumption

No adverse effect on water consumption was detected.

Post Mortem Studies

All females treated with 600 mg/kg bw/day exhibited thickening and/or sloughing of the non-glandular region of the stomach. Isolated occurrences of yellow colored contents in the stomach and raised white patches on the non-glandular region of the stomach were also apparent.

No macroscopic abnormalities considered to be related to treatment with the test item were noted in animals treated with 200 or 50 mg/kg bw/day.

Litter Data and Litter Placental and Fetal Weights

The number of implantations and subsequent embryofetal survival were unaffected by maternal treatment at 50, 200 or 600 mg/kg bw/day. At 600 mg/kg bw/day, mean fetal weights for both sexes were lower than control, resulting in lower mean litter weights. Placental weights were also reduced at this dose level.

No toxicologically significant effects were detected at 200 or 50 mg/kg bw/day.

Fetal Examination

External Findings

An increased incidence of small fetuses was evident from females treated with 600 mg/kg bw/day which resulted in a statistically significant increase in total number of fetuses with external findings. No such findings were evident in litters from females treated with 200 or 50 mg/kg bw/day.

Detailed Visceral Examinations

Visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 50, 200 or 600 mg/kg bw/day.

Detailed Skeletal Examination

At 600 mg/kg bw/day, there was clear effect of treatment on a large number of ossification parameters affecting most regions of the skeleton, with the number of fetuses/litters affected being increased compared with control (areas not ossified or showing incomplete ossification). Decreases were also noted in the number of fetuses/litters showing ossified areas. No treatment-related adverse effects were detected in the type and incidence of skeletal findings in fetuses from females treated with 200 or 50 mg/kg bw/day.

Conclusion

The oral (gavage) administration of 1,1,3,3-tetramethylbutyl hydroperoxide (CAS#5809-08-5) to pregnant rats from gestation Days 5 to 19, at dose levels of 50, 200 or 600 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an effect on food consumption at 600 mg/kg bw/day. Slight effects on body weight development were noted at 200 mg/kg bw/day, however, these were considered not to be toxicologically significant. No similar effects were apparent at 50 mg/kg bw/day. Consequently, 200 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female and 50 mg/kg bw/day was considered to represent the No Observed Effect Level (NOEL) for the pregnant female.

In-uterosurvival of the developing conceptus was unaffected by maternal treatment with 600 mg/kg bw/day, although reduced fetal and placental weights and an increased incidence of skeletal findings indicated an adverse effect on fetal growth. No changes in the measured fetal parameters or embryofetal development were detected at 200 or 50 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 200 mg/kg bw/day.