Diisobutyl hexahydrophthalate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted according to internationally recognised test methods.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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

Sex:

male/female

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.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Substance is poorly soluble in water
- Concentration in vehicle: As required to achieve nominal dose, up to 25% w/v
- Amount of vehicle (if gavage): 2mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days (until sperm detected in vagina).
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): singly
- Any other deviations from standard protocol: None reported

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%).

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 during treatment period

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 100, 300and 1000 mg/kg/day
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

Positive control:

No

Examinations

Parental animals: Observations and 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:

Oestrous cyclicity (parental animals):

Examined pre-dose and during mating period

Sperm parameters (parental animals):

Parameters examined in P males/group : yes
- testis weight and microscopic pathology, epididymis weight and microscopic pathology

Litter observations:

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.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following approximately 6 weeks of treatment
- Maternal animals: All surviving animals day 4 post-partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations of all organs & including the cervical, thoracic, and abdominal viscera. Pups: dead pups examined internally and externally. Live pups examined externally and sex confirmed by gonadal inspection..

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.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination): Yes

GROSS NECROPSY
- Gross necropsy of dead pups consisted of external and internal examinations. Examination of live pups was limited to exernal examination.

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

Reproductive indices:

Copulation Index: No. of pairs with successful copulation/no. of pairs mated x 100
Fertility Index: No of males inducing pregnancy/No. of males paired x 100 and No. pregnant females/no. of pairs with successful copulation x 100

Offspring viability indices:

Pre-birth loss: (No. visible implantations - total litter size at birth)/No. visible implantations x 100
Pup loss at birth: (total litter size - live litter size)/total litter size x 100
Cumulative pup loss: (total litter size at birth - live litter size Day 4)/total litter size at birth x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation noted in animals treated at 1000 mg/kg following dosing

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No mortality occurred in the study. Clinical observations for neurotoxicity assessment (removal of animals from the home cage and open arena) did not reveal changes attributable to the tested substance. Salivation was the most relevant clinical sign, observed approximately 1 hour after dosing in high dose (1000 mg/kg b.w./day) main and recovery animals during the treatment phase of the study. This finding disappeared during the 2 week recovery period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Body weight and body weight gain were unaffected by treatment. Food consumption was unaffected by treatment.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): Measurements of oestrous cycle did not show differences between treated and control groups

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): See histopathology, below.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Measurements of oestrous cycle, copulation plugs, copulatory index and fertility index did not show differences between treated and control groups. An increase in pre-coital interval was noted in 2 females dosed at 1000 mg/kg/day. Due to the low incidence, this finding was not considered to be of toxicological significance. No significant differences were observed in the number of implantation, corpora lutea, pre-implantation loss, total litter size, pre-birth loss and gestation length between control and treated groups.

ORGAN WEIGHTS (PARENTAL ANIMALS): A statistically significant decrease was noted in the absolute thymus weight of females treated at 1000 mg/kg/day (approximately 28%). A slight, statistically significant, increase was also noted in the relative kidney weight of males treated at 300 and 1000 mg/kg/day (approximately 10%) and in the relative kidney and liver weights of females of the recovery group treated at 1000 mg/kg/day (approximately 7%).

GROSS PATHOLOGY (PARENTAL ANIMALS): No treatment-related changes were noted in treated animals, both in main and recovery groups. Those changes that were observed were regarded as being incidental, having a comparable incidence in control and treated groups, and/or characteristically seen in untreated Sprague Dawley rats of the same age.

HISTOPATHOLOGY (PARENTAL ANIMALS): No treatment-related changes were noted in randomly selected animals from the control group and high dose (1000 mg/kg/day) group. Those changes that were observed were regarded as being incidental, having a comparable incidence in control and treated groups, and/or characteristically seen in untreated Sprague Dawley rats of the same age. Examples of such minimal, spontaneous, age-related changes included hepatic and heart inflammatory cell foci. In particular, two female rats treated at 1000 mg/kg/day exhibited mild atrophy of the thymic cortex. As this kind of change is sporadically seen in untreated female rats it was not considered as related to treatment.
A detailed qualitative evaluation of testes was performed on randomly selected control and high dose (1000 mg/kg/day) males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify potential treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. PAS-H stained sections were used to identify the spermatogenic stages. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. Regular layering in the germinal epithelium was noted

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of effects on reproductive performance or sex organs

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING): No effects

CLINICAL SIGNS (OFFSPRING): No effects

BODY WEIGHT (OFFSPRING): No effects

GROSS PATHOLOGY (OFFSPRING): No effects

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of effects on pup weight; sex ratio; survival index; viability index

Overall reproductive toxicity

Reproductive 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:

A repeated-dose toxicity combined with a reproductive/developmental toxicity screen conducted according to OECD Test Guideline No 422 found 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 for both parent animals and offspring, this being the highest dose level investigated.

Executive summary:

A repeated-dose toxicity combined with a reproductive/developmental toxicity screen conducted according to OECD Test Guideline No 422 found 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 for both parent animals and offspring, this being the highest dose level investigated.