Diisopentyl ether

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study following GLPs

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, 2002) and US EPA Guideline OPPTS 870.3650 (2000)

Deviations:

yes

Remarks:

The study integrity was not adversely affected by the deviations

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diisopentyl ether

EC Number:

208-857-4

EC Name:

Diisopentyl ether

Cas Number:

544-01-4

Molecular formula:

C10H22O

IUPAC Name:

3-methyl-1-(3-methylbutoxy)butane

Details on test material:

- Name of test material (as cited in study report): Diisopentyl ether
- Analytical purity: 99.2%
- Lot/batch No.: TAP 645
- Storage condition of test material: 2-8 °C

Test animals

Species:

rat

Strain:

other: Crl:Wl (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: Males 277 g, Females 198 g
- Fasting period before study: Overnight
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm)
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 1 hour prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for
specific gravity of the vehicle and test substance. No correction was made for the purity of the test substance. Solutions were stored at ambient temperature.

VEHICLE
- Justification for use and choice of vehicle: Corn oil, specific gravity 0.92 (Fagron, Nieuwerkerk a/d IJssel, The Netherlands). Corn oil was selected based on trial formulations performed at NOTOX.
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (12 April 2012), according to a validated method (NOTOX project 499367). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 2 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: one female/one male
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post-coitum
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

Once daily

Duration of test:

Parental males were necropsied at completion of mating period (minimum 28 days of treatment), females which delivered were necropsied in lactation days 5-7. Non-pregnant females were necropsied in day 26 post-coitum. Pups were examined (necropsied) in lactation days 5-7.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 animals/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In order to set the dose levels for the main study, a dose range finding study was performed. Groups of 3 females (11 weeks old) were dosed at 500 or 1000 mg/kg/day for 15 days by oral gavage. In both groups, the food consumption was slightly reduced for the first 10 days. No clinical signs or effects on body weights were observed. In the 1000 mg/kg bw group an increase in the liver weights was observed. Based on the results of this range finding study, dose levels for the main study were: 100, 300 and 1000 mg/kg body weight.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately after each dosing, once prior to start of treatment and at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: Parental males were necropsied at completion of mating period (minimum 28 days of treatment), females which delivered were necropsied in lactation days 5-7. Non-pregnant females were necropsied post-coitum days 25-27 (females with evidence of mating) or 21 days after the last day of the mating period (females without evidence of mating).
- Organs examined: Table 1 and Table 2

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: No data
- Number of late resorptions: No data

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

Indices:

Percentage live males at first litter check: Number of live male pups at first litter check/Number of live pups at first litter check x 100
Percentage live females at first litter check: Number of live female pups at first litter check/Number of live pups at first litter check x 100
Percentage of postnatal loss days 0-4 of lactation: Number of dead pups on day 4 of lactation/Number of live pups at first litter check x 100
Viability index (%): Number of live pups on day 4 of lactation/Number of pups born alive x 100

Historical control data:

No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Gestation
Gestation index and duration of gestation were unaffected by treatment.

Parturition/Maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Early postnatal pup development
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal toxicologically significant treatment-related findings.

Mortality
Three pups of the control group, nine pups at 100 mg/kg, one pup at 300 mg/kg and two pups at 1000 mg/kg were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. At the low dose group, the nine dead pups at first litter check were all from one litter (no.
55); this dam was euthanized due to bad health on Day 1 of lactation. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Clinical signs
Incidental clinical symptoms of pups consisted of wound in the genital region, blue spot on the nose and swollen lower lip. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Body weights
At 1000 mg/kg, slightly lower body weights were noted for pups on Day 1 of lactation (only statistically significant for female pups and pups combined). This was not considered toxicologically relevant as Day 4 body weight measurements were not statistically significantly different from the controls and body weight gain over these days was similar (51% for the control group and 52% for Group 4). Body weights of pups at 100 and 300 mg/kg were comparable to the concurrent control values.

Macroscopy
Incidental macroscopic findings of pups that were found dead included beginning autolysis, absence of milk in the stomach and/or cannibalism. One surviving pup showed swollen lower lip. The nature and incidence of these findings remained within the range considered normal for pups of this age, and
were therefore considered to be of no toxicological relevance. There were two litters (no. 45 of the control group and no. 75 of the high dose group) of which all pups showed absence of milk in the stomach. For litter no. 45, this was considered a chance finding as it concerned a litter from the control group. For litter no. 75, this was due to an error as the pups were separated from their mother one day before necropsy (reported as protocol deviation).

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Refer to "details on maternal toxic effects"

Remarks:

Refer to "details on maternal toxic effects"

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:

dose level:

Effect level:

ca. 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Refer to "details on Embryotoxic /teragenic effects"

Remarks on result:

other: Refer to "details on Embryotoxic /teragenic effects"

Remarks:

Refer to "details on Embryotoxic /teragenic effects"

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Analysis of Dose Preparations

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 2 hours.

Applicant's summary and conclusion

Conclusions:

No reproduction/developmental toxicity was observed at any dose level. Based on these results, a reproduction and developmental No Observed Adverse Effect Level. (NOAEL) of 1000 mg/kg was derived.

Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted with diisopentyl ether in rats by oral gavage.

The study was based following guidelines from Organisation of Economic Co-operation and Development Guidelines (OECD) for testing of Chemicals Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (1996) and The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (2000).

Based on the results of a 15-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg.

After acclimatisation, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

The following parameters were evaluated in the parental animals: mortality/viability, clinical signs, functional observations, body weights, food consumption, reproduction/developmental parameters, observations pups, clinical pathology, macroscopy, organ weights, and histopathology. Pups were examined for viability, clinical signs, and body weights were determined. All pups were sexed and descriptions of all external abnormalities were recorded at necropsy. Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability.

No reproduction/developmental toxicity was observed at any dose level. Based on these results, a reproduction and developmental No Observed Adverse Effect Level. (NOAEL) of 1000 mg/kg was derived.

Based on the increased liver weights (mid and high dose females and high dose males) and treatment-related microscopic findings in thyroid (males), thymus (males) and liver (both sexes), a parental NOAEL of 100 mg/kg bw/day was established.