Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

A 28-day repeated dose study and a 90-day repeated dose study in rats with expsoure via the oral route are available. Both studies are reliable (Klimisch 1 studies). In addition, a 28-day repeated dose study (Klimisch 2 study, study only included males) and a 90-day study (Klimisch 1 study) via the inhalation route are available.


No data are available for repeated exposure via the dermal route.


 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31st May 2016 to 15th September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
21 September 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 72528
- Expiration date of the lot/batch: 15 February 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: Stability for test formulation with vehicle under test conditions was investigated and proved for a 10 day retention time.
- Solubility and stability of the test substance in the solvent/vehicle: Stability for test formulation with vehicle under test conditions was investigated and proved for a 10 day retention time.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was weighed into a tared plastic vial on a precision balance. The dose formulations were prepared by adding the required volume of aqua ad iniectabilia (sterile water) and further vortexing it for 2-3 minutes.
The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and due to previous toxicological tests also using sterile water as vehicle. The test item formulation was prepared once in 10 days.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration. The vehicle was also used as control item.
- Preliminary purification step (if any): None

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Species/strain: Sprague-Dawley rats, CD rats Crl:CD(SD) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Species/strain: Sprague-Dawley rats, CD rats Crl:CD(SD) (Full Barrier)
- Number and sex of animals: 80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks old
- Weight at study initiation: males: 191 – 232 g (mean: 210 g, ± 20% = 168 – 252 g); females: 146 – 177 g (mean: 160 g, ± 20% = 128 – 192 g)
- Fasting period before study: None
- Housing: Full barrier in an air-conditioned room. The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: Adequate acclimatisation period (at least 5 days)

DETAILS OF FOOD AND WATER QUALITY:
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, 12 hours light, 12 hours dark

IN-LIFE DATES: 7 June 2016 - 15 September 2016
Route of administration:
oral: gavage
Details on route of administration:
The test item or vehicle was administered as a single daily dose by oral gavage at a volume of 5 ml/kg body weight. The individual dosing volume for each animal was calculated based on the most recent body weight measured.
Vehicle:
water
Remarks:
aqua ad iniectabilia (sterile water)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a precision balance. The dose formulations were prepared by adding the required volume of aqua ad iniectabilia (sterile water) and further vortexing it for 2-3 minutes.
The test item formulation was prepared once in 10 days.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration. The vehicle was also used as control item.

VEHICLE
- Concentration in vehicle:
Control: 0 mg/ml
Low dose: 10 mg/ml
Mid dose: 50 mg/ml
High dose: 200 mg/ml
- Amount of vehicle (if gavage): The application volume for all groups was 5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
- Lot/batch no. (if required): 605070
- Purity: Not reported

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation samples from all dose groups were retained in weeks 1, 5, 9 and 13 of the treatment period. Approximately 5 ml of each sample was retained in duplicate (sample A and sample B). Samples A were analysed on the same day (samples from week 1, 5 and last) or after 6 days (samples from week 9). Samples B were retained as backup until the analysis of samples A had been performed. Samples were kept at -15 to -35°C for three months following the date on which the final phase report was audited by the Quality Assurance Unit.

Stability was investigated and proven for 10 days retention time.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Single dose via gavage daily.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Nominal doses confirmed by chemical analysis.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Nominal doses confirmed by chemical analysis.
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Nominal doses confirmed by chemical analysis.
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Nominal doses confirmed by chemical analysis.
No. of animals per sex per dose:
80 animals (40 males and 40 females; 10 male and 10 female animals per group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Justification for the Selection of the Test System:
The rat is a widely used rodent species in toxicological studies and acceptable to regular authorities.
This study provides information on the possible health hazards which could arise from repeated exposure over a period of 90 days.

- Dose selection rationale: Doses were selected based on previous results from a 28-day repeat dose study. 3 dose groups were selected (LD = low dose, MD = medium dose, HD = high dose). The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
- Rationale for animal assignment (if not random): Before the first administration all animals were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 9.4.0 software).
- Section schedule rationale (if not random): The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 90 days. 10 animals per gender and group were subjected to necropsy one day after the last administration (end of treatment period).
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once before the first administration and once a week thereafter.
- Cage side observations checked in table [No.?] were included. Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed for clinical signs during the entire treatment period of 90 days. General clinical observations were made once a day, approximately at the same time each day after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period.
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. Mean body weights are also be presented as figures.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food consumption was measured weekly during the treatment period.
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. Mean body weights are also be presented as figures.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period.
- Dose groups that were examined: All animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: All sacrificed animals
- Parameters checked: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), and large unstained cells (Luc).

BLOOD COAGULATION
- Time schedule for collection of blood: Coagulation parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.
- Animals fasted: Yes
- How many animals: All sacrificed animals
- Parameters checked: prothrombin time (PT); activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.
- Animals fasted: Yes
- How many animals: All sacrificed animals
- Parameters checked: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K).

URINALYSIS: Yes
- Time schedule for collection of urine: A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: No
- Parameters checked: The following parameters were measured using qualitative indicators
(Urine, Stripes, Henry Schein).
specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL), erythroctes (Ery), leukocytes (Leu).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first exposure and once in the last week of exposure multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests.
- Dose groups that were examined: These tests were conducted in all animals.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
One day after the last administration (study day 91) all surviving animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. The wet weight of the organs of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead were not recorded.

Following organs were weighed: liver; uterus with cervix; kidneys; thymus; adrenals; thyroid/ parathyroid glands; testes; spleen; epididymides; brain; prostate, seminal vesicles and coagulating glands; pituitary gland; ovaries; and heart.

HISTOPATHOLOGY: Yes

Tissues included: adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum , kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin , spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid gland including parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina.
Statistics:
Body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next.
The relative organ weights were calculated in relation to the brain weight and in relation to the body weight (measured at necropsy) and are presented as percentage.
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 is considered as statistically significant).
Clinical signs:
no effects observed
Description (incidence and severity):
The weekly detailed clinical examination showed no significant differences in test-item groups compared to the corresponding control group. Effects such as alopecia were observed in all dose groups including the controls which indicates that this is not a treatment related effect. Effects such as moving bedding and salivation were also observed. Salivation just before the dose administration was considered to be a conditional reflex which persisted even after the dose administration. The moving bedding immediately after the dose administration was considered to be due to the local effect caused by test-item gavage.
Mortality:
no mortality observed
Description (incidence):
There was no mortality due to test-item toxicity. Two males and one female died to gavaging error.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In males and females, there were no effects on body weight in test-item groups compared to the control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In males and females, the food consumption from day 1 to day 90 was not affected in test-item groups compared to the control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The high dose group (males) showed a statistically significant increased mean lymphocyte count and lower mean neutrophil count when compared to controls. In the high dose female group statistically significantly lower mean reticulocyte and higher prothrombin time were reported when compared to controls. The differences between the highest dose groups and the controls are only marginally different for these parameters. Considering no other adverse effects in the study, the findings were considered to be of minor toxicological relevance

Other effects such as significantly lower mean haemoglobin levels in high and mid dose males (16.21 g/dl and 16.36 g/dl, respectively), lower red blood cell counts in mid dose males and lower basophil levels in the low dose males were observed. However there were no clear dose-response relationships for these effects and therefore are not considered treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the high dose female group, statistically significant higher mean glucose and potassium levels were observed when compared to controls. The differences between the highest dose groups and the controls are only marginally different for these parameters. Considering no other adverse effects in the study, the findings were considered to be of minor toxicological relevance

Other effects such as significantly lower mean aspartate-aminotransferase (ASAT) in all dose groups in females and lower alanine aminotransferase (ALAT) in females from the low and mid dose groups were observed but are not toxicologically relevant.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urine parameters showed no significant effects in the animals of the test-item groups compared to the control.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The neurological assessment revealed no significant differences in functional neurobehavioural parameters between the controls and treated groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
In males and females, there were no statistically significant differences in the absolute and relative (to brain and body weight) organ weights measured at necropsy.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All gross lesions recorded at necropsy were considered to be within the range of normal historical control values.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mid and high dose females showed an increase of inflammatory cell foci in the liver. In the absence of necrosis, Kupffer’s cell proliferation, apoptosis, fibrosis, alteration in liver function and no significant increase in alanine aminotransferase (ALAT) or aspartate-aminotransferase (ASAT), this finding is deemed to be of a non-adverse nature.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: blood parameters
Organ:
other: blood system
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes
Conclusions:
A NOAEL of 250 mg/kg bw/day was derived based on statistically significant increased mean lymphocyte count and lower mean neutrophil count in high dose males, lower mean reticulocyte, higher prothrombin time, and higher glucose and potassium levels in high dose females.
Executive summary:

On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with MMB (3-Methoxy-3-Methyl-1-Butanol) in male and femaleSprague-Dawleyrats, with dose levels of 0, 50, 250, and 1000 mg/kg body weight/ day the following conclusions can be made:

Treatment with the test item MMB (Methoxy-3-Methyl-1-Butanol) induced an increase of inflammatory cell foci in the liver of medium and high dose females. However, in the absence of necrosis, Kupffer’s cell proliferation, apoptosis, fibrosis, alteration in liver function and no significant increase in alanine aminotransferase (ALAT) or aspartate-aminotransferase (ASAT), this finding is deemed to be of a non-adverse nature.

The haematological data showed a statistically significantly higher mean lymphocyte in the male high dose group (85.47%) compared to the control (79.59%), statistically significantly lower mean neutrophil in the male high dose group (10.69%) compared to control (15.95%), a statistically significantly lower mean reticulocyte in the female high dose group (1.63%) compared to the control (2.24 %) and a statistically significantly higher mean prothrombin time value in the female high dose group (22.87 Sec) compared to the control (19.82 Sec). The clinical biochemistry data showed a statistically significantly higher mean glucose in female high dose (10.309 mmol/L) compared to the control (8.676mmol/L) and statistically significantly higher mean potassium in female high dose group (4.514 mmol/L) compared to the control (3.843 mmol/L). The differences between the highest dose groups and the controls are only marginally different for these parameters, but they are statistically significant and indicate a toxicological response to the test item and therefore are considered significant effects.

Based on the results of haematological and clinical biochemistry parameters, the LOAEL for the systemic toxicity is 1000 mg/kg bw/ day and the NOAEL is 250 mg/kg bw/ day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two reliable studies are available.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 4, 2019 - October 2, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was performed for worker safety requirements.
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Storage conditions: Room temperature (actual range: 14.9°C to 22.8°C), dark place, airtight container, filled with nitrogen
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
This strain is widely used in toxicity studies using rodents, and there are abundant historical data.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at start of exposure: 8 weeks old.
- Body weight range at start of exposure: Males: 269.0 to 311.3 g, females: 181.5 to 219.7 g.
- Housing: Two animals (same sex) /cage.
Cages: Stainless-steel wire mesh hanging cage (W 390 × D 285 × H 180 mm).
Racks: Stainless steel racks.

- Diet: Pellet feed for experimental animals (ad libitum) except for the following periods:
· During exposure.
· During fresh urine collection.
· Leading up to scheduled necropsy from the evening on the day before necropsy.

- Water: Tap water filtered through a 5-µm pore size filter followed by irradiation with ultraviolet
light. Provided ad libitum, except no water was provided during inhalation exposure or fresh urine collection.

- Acclimation period: 6 days.

DETAILS OF FOOD AND WATER QUALITY: Analysis results were obtained from the food supplier, and then levels of contaminants such as residual pesticides in the used lots of food were confirmed to meet the criteria specified in the SOP of the test facility. Water quality was periodically (twice a year) analyzed by an external agency. The values obtained from the analysis were confirmed to meet the criteria specified in the SOP of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.1°C to 23.5°C.
- Humidity: 37.3% to 69.5%
- Air changes: Six to 20 times/hr, all fresh air.
- Photoperiod: Twelve hr/day.
Route of administration:
inhalation: mist
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: A flow past nose-only inhalation exposure chamber (hereinafter referred to as “chamber”). The chamber is constructed from stackable tiers, which had 16 exposure ports per tier. This study used a chamber constructed from two tiers.

- Method of holding animals in test chamber: Animals individually held in the restraint tube.

- System of generating particulates/aerosols: The test substance was converted into a mist containing the test substance in vapor-form using a double-fluid nebulizer (NB-2N: Shibata Scientific Technology Ltd.) by supplying pressurized air and the test substance at a prescribed flow rate.

- Temperature in air chamber: The generated mist was passed through a heat exchanger with a set temperature of 40°C to prepare a test atmosphere containing the vaporized test substance at a specified concentration. The test atmosphere was continuously supplied to the chamber for exposure to animals.

- Air flow rate: The flow rate of the test atmosphere supply to the chamber was set at 16 L/min/tier (permissible range: 16.0 to 22.0 L/min/tier). The flow rate through the chamber exhaust was set at approximately 10% lower than the test atmosphere supply, because the pressure inside the chamber must be positive to ensure reliable exposure.

TEST ATMOSPHERE
- Brief description of analytical method used:
* Syringe: 1 mL gas tight syringe (#1001)
* Sampling bag: 1.56 L inner volume (actual inner volume, Smart bag)
* Vacuum sampling container: A container for sampling the test atmosphere into the constituent sampling bag by reducing the pressure inside (made in-house).
* Air pump: General purpose pump.

- Samples taken from: Exposure port of the chamber.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nominal concentration was calculated from the amount of test substance supplied to the chamber and the air volume passed through the chamber. Measurement of exposure concentration and determination of concentration stability was done using a validated analytical method (gas chromatography).

Sampling position: Exposure port of the chamber.
Sampling time point:
30 minutes, 3 hours, and 5 hours 30 minutes after the start of exposure on the day of the start of exposure (males), and at 7-days intervals thereafter 30 minutes after the start of exposure on other days.
Number of sample: 1 sample/time point.
Sampling method: The sampling bag was placed into the vacuum sampling container, and connected to the exposure port. The test atmosphere as an analytical sample was sampled into the sampling bag by reducing the pressure inside the container using an air pump.

One analysis per sample was performed. The concentration of the test substance was calculated from the peak area derived from the test substance and calibration curve (minimum unit: 0.01 mg/L).

Time of measurement:
Start of test atmosphere generation, start of exposure, and once an hour thereafter until completion of exposure, and termination of test atmosphere generation.
Duration of treatment / exposure:
13 weeks.
Frequency of treatment:
Inhalation exposure was conducted once a day for 6 hours, 5 days a week, for 13 weeks.
Dose / conc.:
0 mg/L air
Remarks:
Control
Dose / conc.:
0.25 mg/L air (nominal)
Remarks:
The measured concentration was 0.27 mg/L.
Dose / conc.:
1 mg/L air (nominal)
Remarks:
The measured concentration was 1.04 mg/L.
Dose / conc.:
2 mg/L air (nominal)
Remarks:
The measured concentration was 2.07 mg/L.
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes
Details on study design:
- Dose selection rationale: The exposure concentration was set to obtain information to determine whether or not the test substance falls into Category 2 of the GHS classification criteria for specific target organ toxicity (repeated exposure). The guidance values for concentrations that cause toxic effects equivalent to GHS Category 2 are greater than 0.02 mg/L and less than 0.2 mg/L for mist or dust, and greater than 0.2 mg/L and less than 1.0 mg/L for vapor.

In a preliminary subacute inhalation toxicity
study (28-day study, study No. B180761), high levels of plasma inorganic phosphorus were observed in males in groups exposed to 1 mg/L or more of the tests substance and high relative kidney to body weight ratio was observed in males and females of the 2 mg/L group. No toxicological changes related to the test substance were observed in the 0.5 mg/L group; however, the possibility of toxic effect could not be eliminated in a 90-day study with a longer exposure period. Based on the above observations, target exposure concentrations needed to obtain information to determine whether or not the test substance falls into the GHS Category 2 classification for specific target organ toxicity (repeated exposure) were set at 0.25 mg/L as the low exposure concentration, and 1 and 2 mg/L as the middle and high exposure concentrations, respectively.


- Animals assignment: The animals were allocated to study groups so as to obtain approximately equivalent mean body weights among the groups using the stratified-by-weight randomization method based on body weight on the allocation day.

- Fasting period before blood sampling for clinical biochemistry: One night. Animals were fasted from the evening on the day before necropsy until the necropsy.
Observations and examinations performed and frequency:
1- Clinical signs:
Daily observations were conducted from the first day of exposure to the day of necropsy.
Exposure day: Twice a day (before and after exposure).
Other days: Once a day.

2- Body weight:
Body weight was measured according to the schedule below. Measurements were conducted before exposure on the day of exposure.

Male: Day 1, Day 6, Day 8, Day 13, Day 15, Day 20, Day 22, Day 27, Day 29, Day 34, Day 36, Day 41, Day 43, Day 48, Day 50, Day 55, Day 57, Day 62, Day 64, Day 69, Day 71, Day 76, Day 78, Day 83, Day 85, Day 90, and Day 91.

Female: Day 1, Day 5, Day 8, Day 12, Day 15, Day 19, Day 22, Day 26, Day 29, Day 33, Day 36, Day 40, Day 43, Day 47, Day 50, Day 54, Day 57, Day 61, Day 64, Day 68, Day 71, Day 75, Day 78, Day 82, Day 85, Day 89, and Day 91.

On the day of the scheduled necropsy (Day 92), body weight was measured to calculate
relative organ weights (relative body weight ratio).

3- Food consumption:
Gross weight of food per cage was measured according to the following schedule:
Day 1, Day 8, Day 15, Day 22, Day 29, Day 36, Day 43, Day 50, Day 57, Day 64, Day 71, Day 78, Day 85, Day 87, and Day 91

Mean daily food consumption per animal housed in the same cage was calculated from the weight difference between each measurement day.

4- Ophthalmological examination:
Schedule:
Pre-exposure: Week -1
Exposure period: Week 13

5- Urinalysis:
Urinalysis was conducted in 6 animals from each group.
Schedule:
Week 13 (Day 85 to Day 86).
Parameters are summarized below.

6- Hematology:
Schedule:
The day of scheduled necropsy (Day 92).
Parameters are summarized below.

7- Blood chemistry:
Schedule:
The day of scheduled necropsy (Day 92).
Parameters are summarized below. (Thyroid hormones were not included).
Sacrifice and pathology:
* Pathological examination:
Examined organs and tissues:
The organs and tissues collected and examined are listed in the file attached in "Overall remarks, attachments".

* Necropsy:
All animals were subjected to necropsy on Day 92.
Animals were subjected to necropsy after euthanization by exsanguination from the abdominal aorta after blood collection.

* Organ weight:
The organs and tissues listed in the file attached in "Overall remarks, attachments". Animals were weighed using an electronic balance (AG204).
Bilateral organs were weighed simultaneously. The pituitary and thyroid/parathyroid were weighed after fixation with formalin. Moreover, relative organ weight (relative body weight ratio) was calculated from body weight measured on the day of necropsy.

* Histopathological examination.
Microscopic examination was conducted on specimens from all animals in the control and high exposure concentration groups, and organs and tissues with gross abnormalities from all animals, which were collected at the end of the exposure period.
Other examinations:
* Examination of bronchoalveolar lavage fluid (BALF): The day of scheduled necropsy (Day 92).


Deviation from the study protocol:
Lung fixation:
The study protocol indicated that the lung was to be perfused with the fixation solution at 25 cmH2O and kept for 15 minutes. The actual duration was 15 to 25 minutes. A duration of 15 minutes was sufficient for lung fixation; therefore, the description in the study protocol was judged to be incorrect. Since sufficient fixation was conducted, it was concluded that this did not affect the reliability of the study.

Volume of BALF retrieval solution:
The study protocol indicated that 3 mL of BALF retrieval solution was to be used for the first sampling and 2 mL for subsequent samplings. There was concern that the volume used in males was too small and may influence the accuracy of the study results; therefore, sampling in females was conducted using 5 mL of retrieval solution. Since similar conditions were established according to sex, it was concluded that these operations did not affect the reliability of the study.
Statistics:
The following statistical analyses were conducted on the obtained data. Safety Study System was used for statistical analyses.
Method:
Numerical data:
Initially, variance was assessed using Bartlett's test (significance level: 5%). When the
variance was homogeneous, multiple comparisons test was conducted using Dunnett's
method. When the variance was heterogeneous, multiple comparisons test was conducted
using the Steel test. Multiple comparisons tests were conducted using a two-tailed test with
significance levels of 1% and 5%.

Examined items:
The items subjected to statistical analyses were as follows:
- Body weight.
- Food consumption.
- Urinalysis (specific gravity, volume, and electrolytes).
- Hematology.
- Blood chemistry.
- Organ weight (absolute and relative organ weights).
- BALF examination results.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Loss of fur on the head was observed in 1 female each in the control and 0.25 mg/L groups,
loss of fur on the back was observed in a male in the 2 mg/L group, and swelling of the ear
was observed in 1 male each in the 0.25 and 1 mg/L groups. Since these findings were
external changes caused by the animals being held in a restraining tube during the nose-only
inhalation exposure, it was judged that these findings were not caused by exposure to the test
substance.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in any of the groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control group and any of the
groups exposed to the test substance during the entire observation period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control group and any of the
groups exposed to the test substance during the entire observation period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Particulate opacity in the lens and corneal opacity were both observed to a slight degree, as
spontaneous changes during the exposure period. No changes were caused by test substance
exposure.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was statistically significantly higher reticulocyte ratio, white blood cell count, and lymphocyte count in males in the 0.25 mg/L group; and higher neutrophil count in males in the 2 mg/L group compared to the control group. In absence of a dose-relationship for the observations in the low dose group, these were considered not related to test item exposure. The change in neutrophil count in the 2 mg/L group was not accompanied by a change in white blood cell count and there was no statistically significant difference in the neutrophil ratio. No similar effect on neutrophils was observed in females. Therefore, the changes in hematology were judged to be without toxicological significance. No effects were observed in females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were statistically significantly lower glucose levels in males in the 2 mg/L group (approximately 10% decrease compared to controls) and higher inorganic phosphorous levels in males in the 1 and 2 mg/L groups compared to the control group (appr. +9% and +11.6% compared to controls for 1 and 1 mg/L groups, resp.). No effects were observed in females.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the examination of fresh urine, there was a tendency toward high levels of occult blood
and red blood cells in urinary sediment in males in the 1 mg/L group. Since there were no
exposure concentration dependencies, the changes were considered incidental and to be
without toxicological significance.

In the examination of 16-hour accumulated urine, there were no statistically significant
differences between the control group and any of the groups exposed to the test substance.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were statistically significantly higher absolute values for the spleen in males in the 0.25 mg/L group compared to the control group. (approximately +25% compared to controls). The high average value was contributed to two individual rats in this dose group. Since there was no exposure concentration dependency, the change was judged to be incidental and without toxicological significance. No other effects were observed in male and female rats.

There were statistically significantly lower values for the submandibular glands weights compared to body weights in males in the 0.25 mg/L group (-11% compared to controls), higher values for the heart in males in the 1 mg/L group (+ 7.7% compared to controls), higher values for the liver in males in the 2 mg/L group (+6% compared to controls), and higher values for the kidney in males in the 1 and 2 mg/L groups (+7% and +8% for the males at 1 and 2 mg/L, respectively, compared to the control group). The changes in the submandibular glands in males in the 0.25 mg/L group and heart in males in the 1 mg/L group did not show exposure concentration dependency. Thus, the changes were judged to be incidental and without toxicological significance. All changes were considered to be limited (<10% change compared to controls) and not accompanied by histopathological effects. Therefore these effects were concluded not toxicologically relevant. No effect on organ weight to body weight ratio were observed in females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes attributable to the test substance.
There were unilateral swelling of the pinna in two animals, impression of the cerebrum in one animal, and loss of fur in 3 animals. These were sporadic changes without exposure concentration dependency; therefore, it was judged that these changes were not attributable to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes attributable to the test substance.
Swelling of the pinna observed at necropsy in two animals was auricular chondropathy. Since dilatation of the cerebral ventricle was observed at the impression of the cerebrum in one animal, it was judged to be hydrocephalus. Since the above findings are incidental and commonly observed changes in rats, it was judged that these findings were not attributable to the test substance. There were no histological abnormalities in the skin, although loss of fur was observed in 3 animals.

In the control and 2 mg/L groups, the following histological changes were observed: focal inflammatory cell infiltration in the myocardium of the heart, mineralization of the arterial wall and alveolar macrophage aggregation in the lung, focal inflammatory cell infiltration in the liver, lobular atrophy in the pancreas, basophilic tubule in the kidney, lymphocyte infiltration in the prostate, presence of ultimobranchial remnants in the thyroid gland, and retinal rosette/folding in the eye. The above findings are incidental changes in rats, and there was no significant difference in the number of animals with these findings between groups. In the 2 mg/L group, the following histological alterations were observed: ectopic pancreatic tissue at the serosa of the glandular stomach, focal hypertrophy in the tubules in the kidney, cyst in the pars distalis of the pituitary gland, and accessory adrenocortical tissue in the adrenal gland. The number of animals found with these findings was one and the findings are non-specific changes in rats. Therefore, the findings in the 2 mg/L group were judged not to be attributable to the test substance.
In the control group, the following histological changes were observed: proliferation of epithelial cells in the thymus, focal necrosis of hepatocytes in the liver, cyst in the medulla and infarct in the cortex in the kidney, luteal cyst in the ovary, cyst and aberrant craniopharyngeal tissue of the pars intermedia in the pituitary gland, and focal inflammatory cell infiltration in the Harderian gland. These findings are non-specific changes in rats.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Examination of BALF:
There were no statistically significant differences between the control group and any of the
groups exposed to the test substance.
Details on results:
In blood chemistry, low glucose levels in males in the 2 mg/L group and high inorganic phosphorous levels in males in the 1 mg/L and 2 mg/L groups were observed. Higher relative weights (relative to body weight) of the liver in males in the 2 mg/L group and the kidney in males in the 1 and 2 mg/L groups were observed. Concerning the low glucose levels, the measured value in the 2 mg/L group was 117.0 mg/dL while the background value from the test facility was 141.04 ± 24.65 mg/dL. Concerning the high inorganic phosphorous levels, the measured values in the 1 and 2 mg/L groups were 7.76 and 7.93 mg/dL, respectively while the background value from the test facility was 7.27 ± 0.54 mg/L. Since the changes in blood chemistry were within the range of mean ± 2 S.D. of the background values, it was judged that the changes did not have toxicological significance. Regarding the changes in relative weights (relative to body weight) of the organs, there were no toxicologically significant changes in the blood chemistry or findings in the histopathological examination related to the changes in the kidney or liver. Moreover, there were no toxicologically significant changes in the urinalysis that were related to the change in the kidney. Therefore, although it was not possible to eliminate the possibility that these changes were caused by test substance exposure, it was judged that the changes were not adverse effects.
Key result
Dose descriptor:
NOAEL
Effect level:
2 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: No adverse effects observed up to and including the highest dose tested.
Critical effects observed:
no

See the "Appendix" attached file in "Overall remarks, attachments".

Conclusions:
Based on the results of a 90-day inhalation study, performed according to OECD guideline 413 and GLP principles, the NOAEL of 3-methoxy-3-methylbutanol was concluded to be 2 mg/L, the highest concentration tested.
Executive summary:

The sub chronic inhalation toxicity of MMB was examined in rats. Crl:CD (SD) rats were exposed to the test substance by nose only inhalation exposure for 6 hours a day, 5 days a week, for 13 weeks at the target exposure concentrations of 0.25, 1, and 2 mg/L. Each study group was composed of 10 males and 10 females. The actual exposure concentrations were 0.27, 1.04, and 2.07 mg/L. There were no remarkable changes in testing conditions such as temperature and humidity to affect the study results. 


No abnormalities were observed in clinical signs. Furthermore, there were no changes attributable to the test substance exposure in body weight, food consumption, ophthalmological observation, hematology, blood chemistry, or examination of bronchoalveolar lavage fluid. No abnormal findings attributable to the test substance exposure were observed in necropsy or histopathological examination.


Based on the results of this study, it was concluded that the no observed adverse effect level (NOAEL) of MMB in a 90 day sub chronic inhalation toxicity study was was 2 mg/L in males and females.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 mg/L
Study duration:
subchronic
Experimental exposure time per week (hours/week):
30
Species:
rat
Quality of whole database:
A reliable sub-chronic study is available (Klimisch 1).

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

In a repeated dose oral toxicity study, Crj:CD(SD)IGS rats (5 animals/sex/dose) were given MMB by gavage at 0 (vehicle: distilled water), 15, 60, 250 or 1000 mg/kg bw/day. The administration period was 28 days and the recovery period was 14 days after administration. There were no MMB-induced changes in general condition, body weight gain, food consumption, hematological findings, necropsy findings and histopathological findings. A decrease in chloride in males and females at 1000 mg/kg bw/day and increases in A/G ratio and inorganic phosphorus in males at 1000 mg/kg bw/day were detected. An increase in relative weight of the kidneys in males at 250 (11%) and 1000 mg/kg bw/day (15%) and in females at 1000 mg/kg bw/day (16%), and an increase in relative weight of the liver in males (10%) and females (13%) at 1000 mg/kg bw/day after the administration period and in males at 1000 mg/kg bw/day (7%) after the recovery period were detected. The NOAELs for repeated dose toxicity were considered to be 60 mg/kg bw/day for males and 250 mg/kg bw/day for females.


As the sub-acute toxicity study (28 days) showed no severe toxicity effects according to the criteria for classifying the substance as R48, a sub-chronic toxicity study (90 days) was required according to REACH Annex IX. Based on this repeated dose oral toxicity study, where Sprague-Dawley rats (10 animals/sex/dose) were tested, with dose levels of 0, 50, 250, and 1000 mg/kg body weight/ day the following conclusions were made:


Treatment with the test item MMB induced an increase of inflammatory cell foci in the liver of medium and high dose females. However, in the absence of necrosis, Kupffer’s cell proliferation, apoptosis, fibrosis, alteration in liver function and no significant increase in alanine aminotransferase (ALAT) or aspartate-aminotransferase (ASAT), this finding is deemed to be of a non-adverse nature.


The haematological data showed a statistically significantly higher mean lymphocyte in the male high dose group (85.47%) compared to the control (79.59%), statistically significantly lower mean neutrophil in the male high dose group (10.69%) compared to control (15.95%), a statistically significantly lower mean reticulocyte in the female high dose group (1.63%) compared to the control (2.24 %) and a statistically significantly higher mean prothrombin time value in the female high dose group (22.87 Sec) compared to the control (19.82 Sec). The clinical biochemistry data showed a statistically significantly higher mean glucose in female high dose (10.309 mmol/L) compared to the control (8.676mmol/L) and statistically significantly higher mean potassium in female high dose group (4.514 mmol/L) compared to the control (3.843 mmol/L). The differences between the highest dose groups and the controls are only marginally different for these parameters, but they are statistically significant and indicate a toxicological response to the test item and therefore are considered significant effects.


Based on the results of haematological and clinical biochemistry parameters, the LOAEL for the systemic toxicity was determined to be 1000 mg/kg bw/ day and the NOAEL 250 mg/kg bw/ day.


 


In a repeated dose inhalation toxicity study, T23 -48:JCL-SD rats (10 males/dose) were whole body exposed to MMB vapour at concentrations of 0, 100, 300 and 500 ppm, 4hr/day, 5 days/week for 4 weeks. There were no MMB-induced clinical signs or effects observed on histopathology. No statistically significant changes in body weight, food or water consumption or urine analysis were observed at any dose level. An increase in GOT was noted at the 100 and 500 ppm dose levels, but not at the 300 ppm dose level. Increases in absolute and relative kidney weights were observed in all treated groups, however the effecst were minimal and not concentration-related. The NOAEC for this repeated dose inhalation toxicity study was considered to be 500 ppm.


The sub chronic inhalation toxicity of MMB was examined in rats. Crl:CD (SD) rats were exposed to the test substance by nose only inhalation exposure for 6 hours a day, 5 days a week, for 13 weeks at the target exposure concentrations of 0.25, 1, and 2 mg/L. Each study group was composed of 10 males and 10 females. The actual exposure concentrations were 0.27, 1.04, and 2.07 mg/L. There were no remarkable changes in testing conditions such as temperature and humidity to affect the study results. 


No abnormalities were observed in clinical signs. Furthermore, there were no changes attributable to the test substance exposure in body weight, food consumption, ophthalmological observation, hematology, blood chemistry, or examination of bronchoalveolar lavage fluid. No abnormal findings attributable to the test substance exposure were observed in necropsy or histopathological examination. Based on the results of this study, it was concluded that the no observed adverse effect level (NOAEL) of MMB in a 90 day sub chronic inhalation toxicity study was was 2 mg/L in males and females.


 


 

Justification for classification or non-classification

3-Methoxy-3 -methylbutan-1-ol is not be classified in accordance to Directive 67/548/EEC for specific target organ toxicity following repeated oral exposure since there was no evidence of significant toxic effects in sub-acute and sub-chronic toxicity studies via the oral and inhalation route.