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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with current test methods and in compliance with GLP regulations

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 422
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Diisobutyl hexahydrophthalate
EC Number:
275-069-5
EC Name:
Diisobutyl hexahydrophthalate
Cas Number:
70969-58-3
Molecular formula:
C16H28O4
IUPAC Name:
bis(2-methylpropyl) cyclohexane-1,2-dicarboxylate
Details on test material:
- Name of test material : DIBE (diisobutylhexahydrophthalate)
- Physical state: Liquid
- Lot/batch No.: T60211/349
- Expiration date of the lot/batch: 2012-12-15
- Storage condition of test material: room temperature, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy SpA
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Males: 388-420 g; Females: 242-277 g
- Fasting period before study: No
- Housing: Polysulphone solid bottom cages, Group caged except for in pairs (1 male/1 female) for mating and females individually caged during gestation
- Diet (e.g. ad libitum): Mucedola 4RF21 pelleted diet available ad libitum
- Water (e.g. ad libitum): Municipal supply tap water available ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Prepared daily by dissolution/suspension in corn oil.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Proposed formulation procedure checked for concentration and homogeneity in the range from 20 to 200 mg/mL to confirm that the method was suitable. All levels were within 90-110% of nominal for concentration and the CV for homogeneity was < 10%. Stability after 24 hours at room temperature was verified in the range from 20 to 200 mg/mL and determined to be within acceptable limits (90-110% of nominal of concentration and CV for homogeneity < 10%).
Samples of the formulations, prepared on Weeks 1 and 6, were analysed to check for homogeneity and concentration. Results were within acceptable limits (90-110% of nominal for concentration and the CV for homogeneity was < 10%).
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until mating observed (4 days/1 oestrus)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
Males: from 14 days before pairing for a total of approximetaly 6 weeks
Females: from 14 days before pairing to day 3 post partum (total of approximately 6 weeks)
Frequency of treatment:
Daily, 7 days/week
Duration of test:
Males: from 14 days before pairing for a total of approximetaly 6 weeks
Females: from 14 days before pairing to day 3 post partum (total of approximately 6 weeks)

Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on data findings from structural analogues of the substance
- Rationale for animal assignment (if not random): Random, stratified body weight

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for signs of ill health and moribund condition
- Cage side observations were included.: Yes - Daily, 2 or 3 times following dose administration

DETAILED CLINICAL OBSERVATIONS: Yes (functional observation battery)
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Weekly; Females: days 0, 7, 14, 20, gestation days 0, 7, 14 and 20, post partum Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes - Weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No - Not applicable as animals dosed by gavage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No - Not applicable - Not a dietary study

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No - Not applicable as animals dosed by gavage

SACRIFICE
- Male animals: All surviving animals following 42 days of treatment
- Maternal animals: All surviving animals day 4 of lactation

GROSS NECROPSY:
Gross necropsy consisted of external and internal examinations of all organs & including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The following tissues were weighed: adrenal glands, brain, epididymides, heart, kidney, liver, ovaries (with oviduct), spleen, testes, thymus.
The following tissues were prepared for microscopic examination:(5 males/5 females examined from high dose and control groups) - adrenal glands, bone marrow, brain, caecum, colon, duodenum, epididymides, heart, ileum, jejunum (including Peyer’s patches, kidneys, liver, Lungs (including mainstem bronchi), lymph nodes (cervical and mesenteric), ovaries with oviducts, pituitary gland, prostate gland, rectum, sciatic nerve, seminal vesicles with coagulating gland, spinal cord (cervical, thoracic and lumbar), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus and cervix, vagina.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: Examined for external abnormalities and sex confirmation by gonadal inspection
- External examinations: Yes - all per litter
- Soft tissue examinations: Yes: - early decedents
- Skeletal examinations: No
- Head examinations: No
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups was assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.
Indices:
Copulation Index: No. of pairs with successful copulation/no. of pairs mated X 100
Fertility Index: No of pregnant females/no. of pairs with successful copulation X 100
Implantation index: No. of implantation sites/no. of corporea lutea X 100
Delivery index: No. of pups born/no. of implantation sites X 100
Gestation index: No. of females with live pups delivered/no. of pregnant females X 100
Nursing index: No. of females nursing live pups/no. of females with normal delivery X 100
Live birth index: No. of live pups at birth/no. of pups at birth X 100
Viability index: No. of live pups on d4/no.of live pups at birth

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No deaths occurred. Temporary increase in salivation after dosing was observed in animals treated at 1000 mg/kg/day.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Decreased body weight gain from day 7 to 14 of gestation observed in females treated at 500 mg/kg/day. No adverse effects on body weights in males. No effects on food consumption in either sex.

ORGAN WEIGHTS (PARENTAL ANIMALS): No effects

GROSS PATHOLOGY (PARENTAL ANIMALS): No effects

HISTOPATHOLOGY (PARENTAL ANIMALS): No effects

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

Results (fetuses)

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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Reproductive performance

Dose level (mg/kg body weight/day

0

100

300

1000

Number of mated pairs

10

10

10

10

Number of pregnant animals

9

10

10

10

Copulation index – males (%)

100

100

80

100

Fertility index – males (%)

90

100

80

100

Copulation index – females (%)

100

100

100

100

Fertility index – females (%)

90

100

100

100

Developmental toxicity parameters

Dose level (mg/kg body weight/day

0

100

300

1000

Number of pregnant animals

9

10

10

10

Number of pregnant animals with live young

9

10

10

10

Gestation length (days)

22.11 ± 0.60

22.10 ± 0.57

22.30 ± 0.67

21.80 ± 0.42

Number of corpora lutea

13.67 ± 5.32

15.80 ± 3.29

15.40 ± 4.17

16.50 ± 1.58

Number of implantation sites

13.33 ± 5.24

15.00 ± 3.02

15.00 ± 3.83

16.50 ± 1.58

Pre-implantation loss (%)

2.09 ± 6.27

4.55 ± 7.64

2.10 ± 4.78

0.00 ± 0.00

Pre-birth loss (%)

7.28 ± 6.40

7.89 ± 5.58

10.17 ± 13.41

7.51 ± 6.29

 

 

 

 

 

Lactation day 0:

 

 

 

 

Number of pups born

12.56 ± 5.22

13.80 ± 2.86

13.80 ± 4.24

15.30 ± 2.06

Number of pups alive

12.56 ± 5.22

13.70 ± 2.95

13.80 ± 4.24

15.30 ± 2.06

Pup weight (g)

7.31 ± 1.21

7.14 ± 0.79

7.37 ± 0.83

6.93 ± 0.37

Sex ratio (% males)

47.07 ± 24.44

49.90 ± 12.47

52.01 ± 13.14

45.75 ± 14.51

 

 

 

 

 

Lactation day 4:

 

 

 

 

Number of live pups

12.22 ± 5.02

13.10 ± 2.64

13.70 ± 4.19

15.30 ± 2.06

Pup weight (g)

10.23 ± 2.58

9.79 ± 1.86

10.32 ± 1.80

9.30 ± 0.74

Sex ratio (% males)

46.61 ± 24.52

49.27 ± 12.38

51.71 ± 13.07

45.75 ± 14.51

Applicant's summary and conclusion

Conclusions:
In a repeat-dose toxicity combined with a screening study of reproductive toxicity conducted in accordance with OECD test methods, no adverse effects on oestrous cycle, copulation, fertility, delivery or lactation and no changes related to gestation index, gestation length, numbers of corpora lutea, implantation sites or implantation index. There were no changes in sex ratio, body weight, viability or morphology of pups. The No Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 1000 mg/kg/day, this being the highest dose level investigated.
Executive summary:

In a repeat-dose toxicity combined with a screening study of reproductive toxicity conducted in accordance with OECD test methods,no adverse effects on oestrous cycle, copulation, fertility, delivery or lactation and no changes related to gestation index, gestation length, numbers of corpora lutea, implantation sites or implantation index. There were no changes in sex ratio, body weight, viability or morphology of pups. The No Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 1000 mg/kg/day, this being the highest dose level investigated.

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