Diisopentyl ether

Endpoint summary

Description of key information

In a combined repeated dose study with the reproduction/developmental toxicity screening test in rats with application via the oral route according to OECD Guideline 422 and GLP, a NOAEL of 100 mg/kg bw/day was established 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),

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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 following Good Laboratory Practices

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

Species:

rat

Strain:

other: Crl:WI (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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral 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).

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

Remarks:

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

Remarks:

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

Remarks:

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

No. of animals per sex per dose:

10 males and 10 females per 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:

None

Observations and examinations performed and frequency:

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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY/CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight with a maximum of 20 hours.
- How many animals: 5 animals/sex/group
- Parameters checked in table No. 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Selected males were tested during week 4 and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling)
- Dose groups that were examined: All groups (5 animals/sex/group)
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex, static righting reflex), grip strength and motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)

The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.
- The preserved organs and tissues of the animal that was killed in extremis (no. 80).
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all animals that failed to mate, conceive, sire or deliver healthy pups: Group 1 Female 46 and Male 6, and Group 2 Female 56 and Male 16 (both females were non-pregnant).

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.
The following additional methods of statistical analysis were used:
The number of corpora lutea was transformed by using 1/x to obtain a normal distribution. This was followed by an ANOVA. The Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Details on results:

CLINICAL SIGNS AND MORTALITY

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 WEIGHT GAIN

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.

FOOD CONSUMPTION

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.

HAEMATOLOGY

Decreased mean corpuscular haemoglobin concentration (MCHC) levels were noted for males at 100, 300 and 1000 mg/kg and females at 1000 mg/kg. Red blood cell counts were lower for high dose females, and mean corpuscular volume (MCV) levels were increased for females at 300 and 1000
mg/kg.
The remaining statistically significant changes (noted for males at 100 mg/kg for red blood cells, haematocrit and mean corpuscular haemoglobin (MCH)) 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.

CLINICAL CHEMISTRY

At 1000 mg/kg, females showed increased levels of alanine aminotransferase (ALAT), cholesterol and calcium. In addition, an increased concentration of alkaline phosphatase (ALP) was noted for one low dose female (no. 54) and four high dose animals (male nos. 32, 34 and female nos. 71, 76). Aspartate aminotransferase (ASAT) was increased for one female (no. 62) at 300 mg/kg.
All other statistically significant changes (cholesterol for 100 mg/kg males, total bilirubin for 100 mg/kg females, inorganic phosphate for 300 mg/kg females) were considered to be of no toxicological significance 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.

NEUROBEHAVIOUR

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.
Motor activity was unaffected for males up to 1000 mg/kg. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.
The motor activity test at the end of treatment revealed decreased counts for females of all dose groups, however without a clear dose related trend and only statistically significant for total counts at the mid dose. As other functional and clinical observations did not show any abnormalities, this finding was considered not to be adverse.

ORGAN WEIGHTS

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

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: NON-NEOPLASTIC

There were treatment-related microscopic findings.

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.

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 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).

Dose descriptor:

NOAEL

Remarks:

Reproduction/Development

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

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.

Conclusions:

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 after repeated doses of diisopentyl ether in Wistar rats.

Executive summary:

Diisopentyl ether was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats with application via the oral route according to OECD Guideline 422 and GLP (De Raaf-Beekhuijzen, 2012).

The test substance was administered as a solution in corn oil orally by gavage to 3 groups of 10 male and 10 femaleWistar rats each, once a day on 7 days per week. 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. 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.An equally sized

negative control group (group 1) received corn oil, the vehicle for the test substance.

The following parameters were evaluated: mortality/viability, clinical signs, functional observations, body weights, food consumption, reproduction/developmental parameters, observations pups, clinical pathology, macroscopy, organ weights and histopathology. Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability.

Treatment-related microscopic findings were noted in the 1000 mg/kg treated rats in the thyroid (males), thymus (females) and liver (both sexes), and in the kidneys (males) of the 100 mg/kg, 300 and 1000 mg/kg treated rats.

Hyperplasia/hypertrophy of the follicular epithelium of the thyroid was recorded for males 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. This finding may reflect an increase in thyroxine production in response to feedback mechanisms as a result of increased turnover of thyroxine by the hypertrophic hepatocytes.

 

Lymphoid atrophy of the thymus was noted in 1/5 (minimal) 100 mg/kg and 3/5 (2 minimal, 1 slight) 1000 mg/kg treated female rats of the scheduled necropsies. At 1000 mg/kg, decreased thymus weights were noted for the females and males (confirmed at macroscopic examination for one female).

 

Hepatocellular centrilobular hypertrophy was noted in all 1000 mg/kg treated rats at low severities (up to slight). Concurrently, increased liver weights were noted for mid and high dose females and high dose males, and liver enzyme levels (ALAT, ASAT, ALP) were increased for several treated animals.

 

Males at all dose groups showed a dose related increase in incidence and severity of hyaline droplets in the kidneys. Hyaline droplets were considered to represent alpha2u-globulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. In addition, increased kidneys weights were noted for males and females at 1000 mg/kg. For the males, this might be related to the occurrence of hyaline droplets. For females, no corroborative findings were noted. Without any other treatment-related microscopic changes in the kidney or other corroborative findings, the presence of hyaline droplets and the slightly increased kidneys weight were considered to be male rat specific and not relevant to humans.

 

The motor activity test at the end of treatment revealed decreased counts for females of all dose groups, however without a clear dose related trend and only statistically significant for total counts at the mid dose. As other functional and clinical observations did not show any abnormalities, this finding was considered not to be adverse.

 

A few changes were noted for haematology and clinical biochemistry parameters (i.e. decreased mean corpuscular haemoglobin concentration, red blood cell counts and increased mean corpuscular volume, cholesterol and calcium levels). These variations were not considered toxicologically relevant as they were slight and without corroborative findings.

 

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A Klimisch 1 OECD 422 reproduction/development toxicity screening study has been conducted with the substance.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Diisopentyl ether was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats with application via the oral route according to OECD Guideline 422 and GLP (De Raaf-Beekhuijzen, 2012).

The test substance was administered as a solution in corn oil orally by gavage to 3 groups of 10 male and 10 femaleWistar rats each, once a day on 7 days per week. 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. 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. An equally sized negative control group (group 1) received corn oil, the vehicle for the test substance.

No treatment-related clinical abnormalities, differences in food consumption and body weight, and changes in haematology or clinical chemistry parameters were observed up to 1000 mg/kg bw. Macroscopic examination at necropsy did not show any treatment-related gross pathology.

Based on 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.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The recent OECD 422 guideline study performed by De Raaf-Beekhuijzen in 2012 was selected as the key study for this endpoint. Based on the results of this study, a NOAEL of 100 mg/kg/day could be established. The reason for selecting this study is that the design includes both the OECD 407 and OECD 421 guidelines requirements and the exposure duration included an additional 2 weeks exposure duration when compared to a standard 4 weeks toxicity study.

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity studies, the test substance does not meet the criteria of the Directive 67/548/EEC and the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and therefore no classification is needed.