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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

short-term repeated dose toxicity: inhalation
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
other: liquid
Details on test material:
Name of test substance: 2-(2-ethoxyethoxy)-2-methylpropane
Test substance No.: 13/0109-2
Batch identification: 0012428678
CAS No.: 51422-54-9
Purity: NMR: 98.5 g/100 g
GC: > 99.9 area-%
water: 0.02 g/100 g
Homogeneity: The test substance was homogeneous by visual inspection.
Date of production: 01 Jun 2014
Physical state/ appearance: Liquid / colorless, clear
Storage conditions: Room temperature
Storage stability: Expiry date: 01 Jun 2016
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test animals


Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
other: conditioned air: about 50% ± 20% relative humidity, 22°C ± 2°C
Analytical verification of doses or concentrations:
Duration of treatment / exposure:
6h/day, males: 32 days and females: 57 days
Frequency of treatment:
Doses / concentrationsopen allclose all
Doses / Concentrations:
75, 250, and 750 mg/m3
nominal conc.
Doses / Concentrations:
81.1, 243.3, and 726.0 mg/m3
analytical conc.
No. of animals per sex per dose:
Control animals:

Results and discussion

Results of examinations

Details on results:
No deaths were recorded throughout the study. One animal (No. 116) was sacrificed on a moribund state on study day 44 (lactation day 4). The female animal No. 116 showed a moderate mucus cell hyperplasia in the cervix and vagina. No other relevant microscopical findings were found that could explain the cause of the moribund state.
During the whole study period (pre-exposure, pre-mating, mating and post-mating) the male animals showed no clinical signs and findings different from normal. No clinical signs of toxicity were observed in female animals during pre-exposure and pre-mating period. However, clinical signs of toxicity was found in a few female animals during the mating, gestation and lactation period:
• Mating period: During the first two mating days, no vaginal opening was noted in one female animal (No. 130) of the test group 2 (250 mg/m³). The vagina opening was present on mating day 3.
• Gestation: One animal (No. 114) of the test group 1 (75 mg/m³) showed high stepping gait and piloerection on gestation day 23.
• Lactation:
1. One animal (No. 102) of the test group 0 (control animal) showed red vaginal discharge on lactation day 1.
2. Animal No. 114 (test group 1, 75 mg/m³) continued showing high stepping gait and piloerection. Moreover, there were not proper maternal care. Moreover, the pups was not nursed properly. These findings were observed on lactation days 0 and 1. With exception of piloerection, the clinical signs were present on lactation day 2. From lactation day 3 onward, this animal did not show any clinical signs of toxicity.
3. In animal No. 116 (test group 1, 75 mg/m³) gasping, stretched position while breathing, pale skin, red nose discharge, severe salivation were noted. As a result of the severe clinical signs of toxicity the animal No. 116 was sacrificed on a moribund state.
• Exposure period after pups were sacrificed: One animal (No. 140, test group 3) showed injury on the dorsal region on study day onward.
The initially missing vaginal opening of animal No. 130 was biological variation and was not treatment-related. The injury of animal No. 140 appeared as mechanical injury and was not likely to be treatment-related. The other clinical signs of toxicity were observed in animals exposed to low concentration (75 mg/m³) of the test substance. Due to the missing concentration-response relationship, these findings were considered incidental. The detailed clinical observations did not reveal any abnormalities the animals exposed to the test substance.

The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0. The mean body weight change of the male animals of the mid concentration (250 mg/m³) was significantly higher than the controls during pre-mating period from day 7 to day 14. This finding is likely because animals spread the food out of the racks. Thus, it is considered not treatment-related.

No substance-related changes of food consumption was observed during the whole study period in both male and female animals.

In female rats of test group 3 (750 mg/m3) red blood cell (RBC) counts, hemoglobin and hematocrit values were decreased and relative reticulocyte counts were increased.

No treatment-related changes among clinical chemistry parameters were observed.
In rats of both sexes of test groups 2 and 3 (250 and 750 mg/m3) total bilirubin levels were lower compared to controls. In absence of a hypoproliferative anemia this change was most probably due to an increased conjugation rate of bilirubin because of liver enzyme induction followed by an accelerated excretion of bilirubin via the bile. This mechanism was regarded as adaptive rather than adverse.
In females of test group 3 (750 mg/m3) inorganic phosphate levels were slightly higher compared to controls. This was the only changed clinical chemistry parameter in these individuals and therefore the alteration was regarded as treatment-related but not adverse (ECETOC Technical Report No. 85, 2002).

On the day of the performance of the Functional Observation Battery, the animals were not exposed to the test substances.
Observations were performed on study day 54 in females and on study day 26 in males.
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
Besides this, the following findings were observed and have to be assessed individually:
Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
Open field observations:
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups.
Quantitative Parameters:
In general no test substance-related impaired parameters were observed in male and female animals of all test groups.
Overall motor activity (summation of all intervals):
No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all dose groups in comparison to the concurrent control group. Single intervals: No abnormalities were detected.

Absolute organ weight
All mean absolute weight parameters showed no significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly increased:
750 mg/m3: heart (116%) and kidneys (120%)

No histopathological correlate was seen in the significantly increased weights of the heart and kidneys, which were therefore regarded as not treatment-related.
All other mean relative weight parameters showed no significant differences when compared to the control group 0.

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

The extramedullary hematopoiesis in the spleen of females was increased at 750 mg/m3. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The stages of spermatogenesis in males of test group 3 (750 mg/m3) were comparable to those of the control males.

Effect levels

Dose descriptor:
Effect level:
250 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values in females, an increased relative reticulocyte counts in females, and an extramedullary hematopoiesis in the spleen of all females (minimal to moderate)

Target system / organ toxicity

Critical effects observed:
Lowest effective dose / conc.:
750 mg/m³ air (nominal)
Treatment related:
Dose response relationship:
Relevant for humans:

Applicant's summary and conclusion

Under the conditions of this OECD 422 combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats the following NOAEC (no observed adverse effect concentration) were determined for 2-(2-ethoxyethoxy)-2-methylpropane after inhalation exposure:
The NOAEC for local toxicity at the respiratory tract was 750 mg/m³ for the F0 females and males because no adverse findings were observed at the respiratory tract up to the tested high concentration. The NOAEC for general, systemic toxicity was 250 mg/m³ for the F0 females and males based on the normochromic normocytic anemia and the associated extramedullary hematopoiesis in the spleen. The NOAEC for reproductive performance and fertility was 750 mg/m³ for the F0 parental rats. The NOAEC for developmental toxicity in the F1 offspring was 750 mg/m³.
Executive summary:

To evaluate the toxicity profile of 2-(2-ethoxyethoxy)-2-methylpropane after inhalation exposure, groups of ten male and ten female Wistar rats (F0 animals) per test group were exposed nose-only to dynamic atmosphere of 2-(2-ethoxyethoxy)-2-methylpropane for 6 hours per day on each day. The duration of treatment covered a 2-week pre-mating and 2-week mating period in both sexes, 3 days post-mating in males, and the entire gestation period of the females. After the lactation period and after necropsy of the pups total all parental females were exposed to the test substance on 8 days. The target concentrations were 75, 250 and 750 mg/m³. A concurrent control group was exposed to conditioned air. For adaptation to the experimental conditions all animals were transferred into chambers identical to those used in the study and exposed to fresh air on two days (6 hours per day) before start of the exposure period. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance exposure and, as a rule, thereafter at weekly intervals. Clinical observation was performed at least three times on exposure days and once a day during the other days. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. During the 4 exposure days after necropsy of the pups the food consumption was determined also. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20,on the day after parturitionpostnatal day [PND] 1) and on PND 4. After the pups were sacrificed, the females that were exposed for further 8 days were weighed once weekly and once on the last exposure day. A functional observational battery (FOB) was performed and motor activity was measured in 5 parental males and females per group. The FOB of the female animals was on study day 54. The male animals were performed at the end of the exposure period on study day 26. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. Clico-chemical and hematological examinations were performed in5 animals per sex and grouptowards the end of the exposure period. A complete necropsy including gross pathological evaluation and weighing of selected organs was performed. Organs and tissues were examined histopathologically as required by the corresponding test guidelines. All F0 parental animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The following test substance-related adverse effects were noted:

Test group 3 (750 mg/m3)

·       Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values in females

·       Increased relative reticulocyte counts in females

·       Spleen: extramedullary hematopoiesis in all females (minimal to moderate)

Test group 2 (250 mg/m3) and Test group 1 (75 mg/m3)

No treatment-related, adverse effects were measured regarding clinical pathology parameters.