Diisopentyl ether

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted as per OECD guideline, following GLPs

Justification for type of information:

Screening study for reproductive toxicity

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

yes

Remarks:

The study integrity was not adversely affected by the deviations

GLP compliance:

yes

Limit test:

no

Justification for study design:

Screening study for reproductive toxicity

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)

Sex:

male/female

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.

Details on mating procedure:

- 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

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).

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

Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks

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.

Positive control:

No

Examinations

Parental animals: Observations and 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.

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

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

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

Parameters examined in male parental generation: testis weight, epididymis weight, spermatogenic staging.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups (day 1 and 4 of lactation), viability (daily), body weights (day 1 and 4 of lactation), clinical signs (daily).
Necropsy: Pups surviving to planned termination were killed by decapitation on lactation Day 5-8. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: All surviving animals at lactation Days 5-8; Females which were non pregnant (No 52, 61, 66, 67, 72 and No 79) at post-coitum Days 25-27 (females with evidence of mating)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranial, thoracic, and abdominal tissues and organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed Table 2.

Postmortem examinations (offspring):

SACRIFICE
- The offspring were sacrificed on lactation Day 5-8.

GROSS NECROPSY
- Gross necropsy consisted of external aexaminations. The stomach was examined for the presence of milk

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.

Reproductive indices:

Mating (%): Number of females mated/Number of females paired x 100
Fertility index (%): Number of pregnant females/Number of females paired x 100
Conception index (%): Number of pregnant females/Number of females mated x 100
Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the biginning of parturition

Offspring viability 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

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

No treatment related mortality occurred during the study period. One female (no. 55) at 100 mg/kg was killed in extremis on Day 1 of lactation. Piloerection and pale appearance were noted one day earlier. She delivered after 23 days of pregnancy, which is slightly above normal. All nine pups were found dead at first litter check; cannibalism was noted for most of them. At macroscopic examination, the dam showed black contents of stomach and cecum, enlarged liver with many grey-white foci, watery-clear fluid in the thoracic cavity and pale discoloration of the whole body. Moderate coagulative necrosis of the liver was considered the main cause of moribundity.

No toxicologically relevant clinical signs of toxicity were noted during the observation period.

Salivation seen after dosing among animals of the 300 and 1000 mg/kg dose groups, with the highest incidence in the high dose group, was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance.
Alopecia was noted for one animal at 300 mg/kg. At the incidence observed, this was considered not toxicologically relevant.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period. The statistically significant changes noted for body weight gain (on Day 1 of mating for Group 2 males and on Day 11 post-coitum for Group 3 females) were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted. The statistically significant increase in absolute food consumption for high dose females on Days 4-7 post-coitum was considered to be of no toxicological relevance as it was only a very slight increase inconsistent over time and remained within the range considered normal for rats of this age and strain.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

Not examined (gavage study)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

Not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

Not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

Fertility and conception indices were decreased when compared to the control group, however without a clear dose response relationship. This was due to one non-pregnant female at 100 mg/kg, three non-pregnant females at 300 mg/kg and two non-pregnant females at 1000 mg/kg, with none in the control group. Consequently, the mean number of corpora lutea were also decreased in all dose groups when compared to the concurrent control level. Mating index, precoital time and number of implantation sites were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)

At 1000 mg/kg, statistically significantly increased liver and kidneys weights (absolute and relative) were noted for both sexes and decreased thymus weights (absolute and relative) were noted for females and males (not statistically significant). In addition, relative liver weights were also increased
for females treated at 300 mg/kg. The statistically significantly increased thymus weight noted for low dose females was considered to be of no toxicological significance as this occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

GROSS PATHOLOGY (PARENTAL ANIMALS)

One female (no. 71) treated at 1000 mg/kg showed reduced size of the thymus; this was considered treatment related.
The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no
toxicological relevance, and included reddish discolouration of the mandibular lymph nodes, pelvic dilation of the kidneys, soft greenish or yellowish nodule at the epididymides, many dark red foci on the thymus, cervix containing black contents, tan discolouration of the clitoral glands, uterus containing fluid, and alopecia.

HISTOPATHOLOGY (PARENTAL ANIMALS)

The following treatment-related microscopic findings were reported:
Kidneys, males:
There was a dose related increase in incidence and severity of hyaline droplets consisting of 3/5 (2 minimal, 1 slight) in the 100 mg/kg, 4/5 (2 minimal, 2 slight) in the 300 mg/kg and 5/5 (1 minimal, 2 slight, 2 moderate) in the 1000 mg/kg treated rats.
Liver, males and females:
Hepatocellular centrilobular hypertrophy was recorded in all selected 1000 mg/kg treated rats. Recorded severities were low (males: 3 minimal and 2 slight, females: 2 minimal and 3 slight).
Thyroid Glands, males:
Hyperplasia/hypertrophy of the follicular epithelium was recorded at minimal degree in 1/5 0 mg/kg, in 1/5 100 mg/kg and in 5/5 300 mg/kg treated rats and at slight degree in 3/5 1000 mg/kg treated rats.
Thymus, females:
Lymphoid atrophy was noted in 1/5 (minimal) 100 mg/kg and 3/5 (2 minimal, 1 slight) 1000 mg/kg treated rats of the scheduled necropsies. The recorded moderate degree of lymphoid atrophy in female 55 (100 mg/kg, killed moribund) was considered to be caused by the bad condition of this dam due to moderate liver necrosis.
Stomach, female:
A slight degree of hyperplasia and a minimal degree of lymphogranulocytic inflammation were noted focal in the forestomach of 1/5 1000 mg/kg treated rats (Group 4). This focal finding at low incidence and severity is considered to be within background findings.

The remainder of microscopic findings recorded, including the requested prostate, were within the normal range of background pathology encountered in Wistar (Han) rats of this age.

Reproductive performance: No abnormalities were seen in the reproductive organs of the rats who failed to sire or deliver healthy pups, which could account for their infertility. Further, the spermatogenic staging profiles were normal for all examined males.

Effect levels (P0)

Key result

Dose descriptor:

dose level:

Effect level:

ca. 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see "details on results (P0)"

Remarks on result:

other: see "details on results (P0)"

Remarks:

see "details on results (P0)"

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)

The gestation index and duration of gestation were similar between controls and all treated groups. Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment

CLINICAL SIGNS (OFFSPRING)

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 WEIGHT (OFFSPRING)

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.

SEXUAL MATURATION (OFFSPRING)

Not examined

ORGAN WEIGHTS (OFFSPRING)

Not examined

GROSS PATHOLOGY (OFFSPRING)

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).

HISTOPATHOLOGY (OFFSPRING)

Not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

other: see "details on results (F1)"

Overall reproductive toxicity

Reproductive 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:

Based on the 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.