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Toxicological information

Toxicity to reproduction

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Administrative data

screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-01-11 to 2013-05-07
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reliable GLP and guideline study.

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
according to guideline
other: EPA OTP 870.3650 (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 / colorless, clear
Details on test material:
- Name of test material (as cited in study report): tert.-Amyl alcohol; 2-Methylbutan-2-ol

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 9 - 10 weeks (supply), 10-11 weeks (start of pre-exposure), 10-11 weeks (start of exposure, day 0)
- Housing: Makrolon cages type M III, 1 animal, Exceptions: During mating: 1 male/1 female per cage, During rearing up to PND 4: 1 dam with her litter
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

- Temperature: 20-24°C
- Humidity (%): humidity 30-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours light (06:00 -18:00 h), 12 hours darkness (18:00 - 06:00 h)

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
nose/head only
Details on exposure:
For adaptation to the exposure conditions the animals were exposed to fresh air under comparable flow conditions in head-nose inhalation systems on several days before start of the exposure period.

During exposure of the animals, exposure mixtures (inhalation atmospheres: test substance in air) were generated continuously in such a way that they are as homogeneous and as of a constant composition as possible.
- Piston metering pumps
- Two-component atomizers

Generation technique:
For each concentration, constant amounts of the substance to be tested were supplied to heated vaporizers by means of metering pumps. The vapors were mixed with streams of conditioned air and passed into the inhalation systems.

Head-nose inhalation systems:
The test atmospheres were passed into the aerodynamic exposure apparatuses (INA 60, V ≈ 90 L, BASF SE) with the supply air. The rats were restrained in exposure tubes, their snouts projecting into the inhalation chamber to inhale the atmosphere.
The exhaust air system connected to the exposure systems was adjusted in such a way that the amount of exhaust air was lower than the supply air (positive pressure). Thus the test atmosphere was not diluted with laboratory air in the breathing zones of the animals.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum period of 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day [GD] 0)
- After successful mating each pregnant female was caged: If sperm are detected, mating of the pair will be discontinued
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Measurement and recording of technical conditions in the exposure systems

In general, the technical parameters were measured and recorded as follows:
The air flow rates of supply and exhaust air, relative humidities and temperatures in the inhalation systems were measured continuously by an automated measuring system and were monitored against preset limits and partially regulated. The generator parameters temperature and compressed air were also be recorded by means of this system.
All these parameters were recorded continuously by an computerized data acquisition and control system.
The pump rate of the dosing pumps were read and recorded once per exposure. The atomizer pressure was measured continuously by manometers and recorded once per exposure.

Nominal concentration
The nominal concentration of the inhalation atmospheres was calculated from the amounts of test substance dosed and air-flow per unit time.

Analytical methods of determination

The constancy of concentrations in the inhalation atmospheres was surveyed continuously with total hydrocarbon analyzers (FID, Testa). As the measurements with FID presened the sum of the hydrocarbon in the air, to confirm the composition of the test atmosphere, the test atmospheres were analyzed once a week by gas chromatography of absorption samples.

Sampling and ananylses of absorption samples

Absorption samples were taken adjacent to the animals noses in order to confirm the identity of the test substance in the atmospheres. For this purpose, absorption vessels were connected in series, filled with appropriate solvent. Using a gas sampling station appropriate volume of atmosphere was drawn through the absorption vessels, which are analyzed by gas chromatography. Sampling frequency: one sample per concentration and week. The control atmosphere was sampled on one day during the exposure period.

Duration of treatment / exposure:
6 hours
Frequency of treatment:
On 7 consecutive days per week for the desired period of time
Details on study schedule:
Number of exposures:
a) 14 days premating
b) up to 14 days mating
c) Sacrifice after a minimum of 28 days after the first application
a) 14 days premating
b) up to 14 days mating
c) during the pregnancy up to and including GD 19 d) after necropsy of the pups total 9 exposures on 9 consecutive days including the day before scheduled killing
Doses / concentrationsopen allclose all
Doses / Concentrations:
0,732, 2561,7316 mg/m³
nominal conc.
Doses / Concentrations:
0,724, 2523, 6663 mg/m³
analytical conc.
No. of animals per sex per dose:
10 male and 10 female rats
Control animals:
Details on study design:
- Dose selection rationale:
Based on available data, on approval by the sponsor, the following concentrations were selected for the present study:
7316 mg/m³ (2.000 ppm), as high concentration causing toxic effects
2561 mg/m³ (700 ppm), as mid concentration,
732 mg/m³ (200 ppm), as low concentration and expected NOAEC
(3.658 mg/m³ equates approximately 1ppm at room temperature and atmosheric pressure)

Test groups
Male and female Wistar rats were randomized according to their weight and allocated to the test groups before the beginning of the administration period. For each neurofunctional test and motor activity measurement, separate randomization lists were created (random selection).
Substitute animals were ordered with the animal supply, which was available for exchange until beginning of exposure. These animals were treated together with the study collective in the pre-exposure period. A health check of individual animals were performed on supply and on exchange.

- After the end of the administration period (at least 28 days) all surviving parental males were sacrificed and examined.
- The parental females were allowed to deliver and rear their pups until PND 4. On PND 4, all pups were sacrificed and examined as soon as possible.
- After PND 4 of the female, which delivered last, all parental females were exposed to the test substance on 9 consecutive days. They were sacrificed on the day after and were examined.


Parental animals: Observations and examinations:
- Time schedule: twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.

- Time schedule: On exposure days, a clinical inspection was performed on each animal at least three times a day (before, during and after exposure). On non-exposure days, a cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. If such signs occur, the animals were examined several times daily. Abnormalities and changes were documented for each animal. The parturition and lactation behavior of the dams were generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis. On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams were inspected in the afternoons in addition to the evaluations in the mornings.

All animals were subjected to detailed clinical observations (including palpation) outside their cages once before the administration period (day 0), and subsequently once per week (as a rule in the morning), by the same trained technicians, whenever possible. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal. The scope of examinations and the scoring of the findings that were observed were based on the current index of findings in Tox-Lims software and includes but was not limited to the following parameters listed:

1. Abnormal behavior in handling
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency)
16. Assessment of the urine discharged during the examination
17. Pupil size

- Time schedule for examinations:
In general, the body weight of the male and female parental animals were determined once a week at the same time of the day (in the morning), if possible. The following exceptions were notable for the female parental animals:
- During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as the males. Body weight data was only reported in the individual tables.
- Females with litter were weighed on the day after parturition (PND1) and on PND 4.
- Females without litter were weighed once a week. The body weight data of these individuals were only reported in the individual tables.
- After weaning (PND 4), females were weighed once a week until sacrifice; body weight data were only reported in the individual tables.
- After the pups were sacrificed the females were exposed for 9 consecutive days. The F0 females were weight once on the first exposure of this exposure period, once on the third and once on the eight exposure. The last body weight determination was on the day of the gross necropsy.

Generally, food consumption was determined once a week for the male and female parental animals.
- Food consumption was determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
- Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
- Food consumption of the females which gave birth to a litter was determined for PND 1-4.
- Food consumption of the females during the 9 exposure days after necropsy of the pups was determined over one week from study day 47 to day 54.

Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

The FOB was carried out in 5 surviving parental males and females per group. For the males the FOB was carried out at the end of the administration period, for the females at the end of the premating period. No inhalation exposure took place on the day of FOB examination for all animals of the respective sex.

Home cage observation
The rats were observed for about 10-30 seconds in their closed home cages. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait abnormalities
6. Other findings

Open field observation
The rats were transferred to a standard arena (50 x 50 cm with sides of 25 cm height) and observed for at least 2 minutes. In addition to abnormalities, the following parameters were examined with particular attention:
1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/ pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Gait abnormalites
15. Activity/ arousal level
16. Feces excreted within 2 minutes (number/ appearance/ consistency)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearings within 2 minutes

Sensory-motoric test/Reflexes
The rats were then removed from the open field and subjected to the following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Auditory startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings

The MA was carried out in 5 surviving parental males and females per group. For the males the MA was carried out at the end of the administration period, for the females at the end of the premating period.
The MA was carried out on the same day as the FOB was performed. The examinations were performed using the TSE-Labmaster System, TSE , Homburg, Germany. For this purpose, the rats were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Four beams were allocated per cage. The number of beam interrupted was counted over 12 intervals for 5 minutes per interval.
Litter observations:
Status (sex, liveborn or stillborn) and number of all pups delivered from the parents were determined as soon as possible after birth. At the same time, the pups were also examined for gross-morphological changes.

In general, a check was made for any dead or moribund pups twice daily on workdays or as a rule, only in the morning on Saturdays, Sundays and public holidays. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.

All live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented for each pup.

The pups were weighed on the day after birth (PND 1) and on PND 4. The body weight determined on PND 1 was also used to determined runts. Those pups whose body weight was 25% below the mean body weight of the control group (separately according to male and female pups) were defined as runts.

On PND 4, the pups were sacrificed under isoflurane anesthesia with CO2. After sacrificed pups were examined externally and eviscerated, and their organs were assessed macroscopically. Pups that die or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death. All pups without any notable findings were discarded after their macroscopic evaluation.
Postmortem examinations (parental animals):
Blood samples were taken from fasted animals by puncturing the retrobulbar venous plexus under Isoflurane anesthesia. Blood sampling and examination were carried out in a randomized sequence.
The parameters listed below were examined in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.

- Hematology
1. Leukocytes
2. Erythrocytes
3. Hemoglobin
4. Hematocrit
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelets
9. Differential blood count
10. Reticulocytes
11. Preparation of blood smears (only evaluated blood smears will be archived)
12. Prothrombine time

- Clinical chemistry
1. Alanine aminotransferase
2. Aspartate aminotransferase
3. Alkaline phosphatase
4. Serum γ-glutamyl transferase
5. Sodium
6. Potassium
7. Chloride
8. Inorg. phosphate
9. Calcium
10. Urea
11. Creatinine
12. Glucose
13. Total bilirubin
14. Total protein
15. Albumin
16. Globulins
17. Triglycerides
18. Cholesterol
19. Bile acid

- Hormones
Additional serum samples were frozen at -80°C for storage. Measurement of T3, T4 and TSH were carried out only if there was an indication for an effect on pituitary-thyroid axis. The determination was triggered based upon alterations of thyroid histopathology. Depending on the results obtained, samples which were not examined, were stored not longer than 1 year after finalization of the report.

The animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and assessed by gross pathology. Animals which died intercurrently or were killed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Epididymides
3. Testes
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Lung
7. Spleen
8. Thymus

The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal glands
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (tracheobronchial, mediastinal and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladde
48. Uterus
49. Vagina

Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the list below:
1. Adrenal glands
2. All gross lesions
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Larynx (3 levels)
17. Lungs (5 lobes)
18. Lymph nodes (tracheobronchial, mediastinal)
19. Lymph nodes (mesenteric)
20. Nasal cavity (4 levels)
21. Ovaries
22. Oviducts
23. Prostate gland
24. Peyer’s patches
25. Rectum
26. Sciatic nerve
27. Seminal vesicles
28. Spinal cord (cervical, thoracic, lumbar)
29. Spleen
30. Stomach (forestomach and glandular stomach)
31. Testes
32. Thymus
33. Thyroid glands
34. Trachea
35. Urinary bladder
36. Uterus
37. Vagina

Special attention was given on stages of spermatogenesis in the male gonads. Animals that died or were sacrificed in a moribund state were processed histotechnically and assessed like control animals. Special stains of individual organs of individual animals wer prepared if required.
Please refer to any other information on material and methods incl. tables
Reproductive indices:
The testes, epididymides and ovaries of animals that died or had to be sacrificed intercurrently were fixed in 4% buffered formaldehyde solution.
The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites.

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

open allclose all
Dose descriptor:
reproduction toxicity
Effect level:
2 561 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: no treatment related, adverse effects
Dose descriptor:
general toxicity
Effect level:
2 561 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: no treatment related, adverse effects

Results: F1 generation

Effect levels (F1)

Dose descriptor:
Effect level:
7 316 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity/teratogenicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

In males and females of test group 3 (7316 mg/m3), several clinical signs (unconsciousness, apathy, piloerection, reduced attention and unsteady gaits) were observed (during and after exposure) indicating narcotic effect of the test substance. In addition, animals showed some unspecific signs as alopecia and reduced fur care indicating bad general condition of the animals. Occasionally, gasping was observed in single animals.Moreover, reduced attention, apathy, reduced fur care, blood in bedding and vaginal discharge were observed in females of test group 3. Additionally, cholesterol values were increased in both sexes and in males of the same test group glucose levels were lower compared to controls. The absolute and relative liver weights were significantly increased in both sexes of test group 2 (2561 mg/m3) and 3. The mean body weights of the test group 3 male and female animals were significantly lower than the control group. The mating index was 100% in all groups including the controls. The fertility index varied between 80 and 100 % and reflect the normal range of biological variation inherent in the strain of rats used for this study. The mean number of implantation sites, post implantation loss and liveborn pups was comparable between all test substance-treated groups and the controls. No effects were observed regarding pup viability, sex ratio and pup body weights. Regarding histopathology, female animals of test group 3 revealed a reduction of vaginal epithelial height and hypertrophy with increase of mucification. 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.

Applicant's summary and conclusion