Registration Dossier

Administrative data

Description of key information

A well reported 28-day oral study in rats, conducted according to a guideline referenced by the Chemical Substances Control Law of Japan and in accordance with GLP, identified a NOAEL value of 60 mg/kg bw/day in male rats and 250 mg/kg bw/day female rats, based on significant increase in relative kidney weight.  Significant increase in relative liver weight was also observed and a NOAEL of 250 mg/kg bw/day for males and females can be determined. A 90-day oral study in rats according to OECD 408 was conducted as requested by REACH. The purpose was also to assess, if the increase in the relative kidney and liver weight is an adverse effect or not. In this study no statistically significant effects were observed concerning organ weight findings including organ / body weight ratios. Thus, the increases in kidney and liver weight can be considered to be not adverse. The no-effect doses derived from the organ weight findings in the old studies have therefore not been further used in the risk assessment.    

 

However, in the sub-chronic study, 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 were observed. This was identified as the most sensitive organ and the critical adverse effect. Based on these results, a LOAEL of 1000 mg/kg bw/day for systemic toxicity and a NOAEL of 250 mg/kg bw day were determined for the substance.

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
Remarks:
Repeated Dose 90-Day Oral Toxicity Study in Rodents
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
Remarks:
This study was undertaken in accordance with OECD Guidelines for Testing of Chemicals, Section 4, No. 408, “Repeated Dose 90 day Oral Toxicity Study in Rodents” adopted 21 September 1998 and to Good Laboratory Practice. All procedures aheared too with no deviations.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Section 4, No. 408, “Repeated Dose 90 day Oral Toxicity Study in Rodents” adopted 21 September 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 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

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
Minimal reporting of test animals and husbandry, of test substance informations, of inhalation chamber, of exposure data and test conditions. It has not been tested on both sexes, just on male.
Principles of method if other than guideline:
test animals only male, no female /
by using a whole-body mode of exposure no deteails of animals’ breathing zone in an inhalation chamber /
minimal reporting of inhalation chamber - volume" of the test animals should not exceed 5% of the chamber volume /
for 6 hours per day on a 5 day per week basis for a period of 4 weeks / no details of housing conditions and feed of the test animals during the exposure time // no data about measured actual chamber concentration during exposure time /minimal reporting of exposure data - no data about nominal and actual concentrations from the test concentrations in the inhalation chamber
minimal reporting of test conditons
Test animals and husbandry
GLP compliance:
no
Species:
rat
Strain:
other: JCL-SD
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 weeks
- Diet (e.g. ad libitum): Pellet chow CE-2 (CLEA Japan, Inc.) ad libitum
- Water (e.g. ad libitum): municipal tap water ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 0.5°C
- Humidity (%): 60 ± 5%

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
not specified
Details on inhalation exposure:
a total of 20 doses within 4 successive weeks.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
none given
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
4 hours/day, five times/week
Remarks:
Doses / Concentrations:
100, 300, 500 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
10 (male)
Control animals:
yes
Details on study design:
Post-exposure period: None
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observation including behavior and general condition was performed on all the
animals daily

BODY WEIGHT: Yes
- Time schedule for examinations: After the experiment initiation, body weight of all animals was determined before inhalation exposure at the rate of three times a week in the first two weeks (every other day), afterwards, twice a week (Tuesday and Friday). Moreover, weight gain index was calculated (the weight at the time of the experiment initiation is based to be 100).

WATER CONSUMPTION and FOOD CONSUMPTION:
- The total weight of each feeder and each water-supply bottle were measured every other day and the difference between last measurement value and latest measurement value was regarded as the daily food or water consumption.

OPHTHALMOSCOPIC EXAMINATION:No data

HAEMATOLOGY and CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after completion of inhalation exposure experiments (for 20 times)
- Anaesthetic used for blood collection: Yes (pentobarbital sodium)
- Animals fasted: No data
- How many animals: all test animals and control animals
- Parameters checked in table [No. 1 - 4] were examined.

URINALYSIS: Yes
- The urine was collected at necropsy and the parameters listed below were examined using Multistix (Ames corporation).
Protein, Glucose, pH, Ketone, Bilirubin, Urobilinogen.

NECROPSY: Yes
- Macroscopical change was examined for thoracic cavity, organs in abdominal cavity at necropsy. Then, each organ was removed and the weight of the organs was measured. The ratio to the body weight (relative weight) was calculated using numerical
formula below.
Relative weight = organ weight / body weight x 100 (%)

NEUROBEHAVIOURAL EXAMINATION: No data

HISTOPATHOLOGICAL EXAMINATION:
- The following organs were collected from the animals immediately after the completion of necropsy, fixed in a neutral phosphate-buffered formalin solution and dehydrated to prepare 4 µm paraffin sections, and a microscopic examination was performed by hematoxylin eosin (HE) stain:
Liver, Kidney, Adrenal gland, Spleen, Heart, Lung, Brain and spinal cord, Testis

Sacrifice and pathology:

HISTOPATHOLOGY: Yes
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant changes
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No statistically significant changes
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No statistically significant changes
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
An increase of GOT in 100 and 500 ppm.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No statistically significant change
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increases of absolute and relative weights of kidney in all treated groups
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No effect related to the test material
Details on results:

CLINICAL SIGNS AND MORTALITY
There were no atypical signs noted throughout the study in any animals.

BODY WEIGHT AND WEIGHT GAIN
All animals gained weight normally and neither delay of weight gain nor weight loss were noted during the study period. No significant difference from the control group was noted in any of the experimental group.

WATER CONSUMPTION and FOOD CONSUMPTION
In both food consumption and water consumption, no significant difference from the control group was noted in any of the experimental group.

HAEMATOLOGY and CLINICAL CHEMISTRY
The GOT activity in 500 ppm and 100 ppm exposure groups were significantly higher than that of control group (500 ppm exposure group was significantly different at p<0.05 (significance at 95% or above), 100 ppm exposure group was significantly different at p<0.01). In 500 ppm exposure group, one animal showed the remarkable high value of the GOT and GPT activity and one animal showed the remarkable high value of the GOT activity only. In other parameter, no significant difference from the control group was noted in any of the experimental group.

URINALYSIS
In all experimental parameter, no significant difference from the control group was noted in any of the experimental group.

ORGAN WEIGHTS
Although significantly higher values of absolute and relative kidney weights were observed in all experimental groups compared with control groups, there was no significant difference among three experimental groups. In other organs, no significant difference from the control group was noted in any of the experimental group.

NECROPSY
In many animals of all groups including control, slight congestion was observed in lung. (Often observed generally. Not the effect of MMB.) However no macroscopical abnormalities were noted in any other organs.

HISTOPATHOLOGY
Liver
Minute focal hepatic necrosis with slight cellular infiltration and/or debris of cell nucleus was noted in 1 animal of 500 ppm and 300 ppm exposure group, respectively, and in 2 animals of 100 ppm exposure group. It was seen in 1-2 places under the membrane and there was no MMB dose relationships in incidence of this finding. Nodule of round cell in sinusoid or Grisson's Capsule was noted in 6 animals of 500 ppm exposure group, in 7 animals of 300 ppm group, 9 animals of 100 ppm group and 9 animals of control group. This finding was considered to be the extramedullary hematopoiesis, therefore it is considered not to be pathological findings.

Kidney
The minute fibrosis of the cortex (just under the membrane) was noted in 2 animals in both of 500 ppm and 300 ppm exposure group, respectively, in 3 animals of 100 ppm exposure group and in 1 animal of the control group. In these areas, slight local fibrosis was noted and dilatation like cysts was also seen in renal tubule included in these area. The focal lymphocyte infiltration in stroma of the renal cortex (slight) was noted in 1 animal in both of 500 ppm and 300 ppm exposure group, respectively. On the other hands, slight lymphocyte infiltration was noted from mucous membrane of renal pelvis to medulla in 1 animal of the control group. However, glomerulus and uriniferous tubule showed no toxicologically relevant change.

Spleen
In many animals of all groups, congestion was noted. With the exception of congestion, no remarkable changes were noted.

Lung
In both of 500 ppm exposure group and control group, a few small hemorrhagic spots in lung parenchyma were noted in 1 animal, respectively.

Others
No remarkable abnormal findings were noted in testes, heart, adrenal gland, brain and spinal cord.

Dose descriptor:
LOAEC
Effect level:
100 ppm
Sex:
male
Dose descriptor:
LOAEC
Effect level:
530 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: based on 5.3 g/l vapour weight calculated from above
Critical effects observed:
not specified

Table 1: Hematological examination/Biochemical examination [500 ppm]

 Number  red blood cell  ehite blood cell  packed cell volume  hemoglobin  serum total protein  GOT GPT   ALP  BUN
   10^4/ mm^3  /mm^3  %  g/dl  g/dl  Karmen  Karmen  King-armstrong  mg/dl
 1  626  7550  42.0  13.1  6.0  -  -  -  -
 2  608  5500  41.5  13.1  5.6  -  -  -  -
 3  591  5750  42.0  13.9  5.4  72.0  27.0  35.1  19.5
 4  706  5150  42.5  13.4  5.5  61.0  23.0  31.7  19.5
 5  647  7450  40.5  13.1  5.4  62.0  22.0  42.0  18.7
 6  543  6900  41.0  12.9  5.3  107.0  37.0  24.8  27.0
 7  569  10200  40.0  12.6  5.1  80.5  40.0  32.8  25.9
 8  583  7100  40.0  12.9  5.4  121.0  88.5  40.7  22.7
 9  623  10200  40.0  13.1  5.4  65.0  27.5  37.8  24.1
 10  577  5750  39.0  12.8  5.0  86.0  44.0  35.0  18.7
 Mean  607  7155  40.9  13.1  5.4  81.8  38.6  35.0  22.0
 S.D.  44  1719  1.1  0.3  0.3  20.6  20.3  5.1  3.2

Table 2: Hematological examination/Biochemical examination [300 ppm]

 Number  red blood cell  ehite blood cell  packed cell volume  hemoglobin  serum total protein  GOT GPT   ALP  BUN
   10^4/ mm^3  /mm^3  %  g/dl  g/dl  Karmen  Karmen  King-armstrong  mg/dl
 11  645  6000  41.0  13.2  6.0  59.0  20.0  35.3  16.9
 12  654  7400  44.0  14.0  6.4  86.0  29.0  41.8  19.5
 13  698  7250  43.0  13.7  5.9  59.5  22.0  43.6  17.0
 14  546  4900  43.5  12.9  5.0  70.0  20.0  31.7  18.7
 15  581  5450  40.5  12.9  5.4  62.0  18.0  42.0  21.4
 16  618  7750  40.5  13.1  5.4  75.0  31.0  21.9  22.5
 17  596  7450  40.0  13.3  5.4  -  -  -  -
 18  614  9250  42.5  13.3  5.4  81.5  37.0  33.0  16.5
 19  584  7050  38.5  12.6  5.0  66.0  30.5  36.3  20.8
 20  611  8750  39.5  13.1  5.0  63.0  26.0  32.8  18.7
 Mean  615  7125  41.3  13.2  5.5  69.1  25.9  35.4  19.1
 S.D.  41  1295  1.7  0.4  0.5  9.2  6.0  6.3  2.0

Table 3: Hematological examination/Biochemical examination [100 ppm]

 Number  red blood cell  ehite blood cell  packed cell volume  hemoglobin  serum total protein  GOT GPT   ALP  BUN
   10^4/ mm^3  /mm^3  %  g/dl  g/dl  Karmen  Karmen  King-armstrong  mg/dl
 21  738  6900  42.5  13.8  6.0  68.0  20.0  41.8  22.0
 22  601  8700  40.5  13.2  6.0  86.0  30.0  42.7  18.2
 23  623  8100  43.5  14.4  6.0  90.0  24.0  35.6  16.9
 24  632  7600  41.0  14.1  5.4  67.5  20.0  31.7  14.4
 25  689  6700  42.5  14.0  5.4  82.5  32.0  32.6  18.7
 26  591  8250  40.5  12.6  5.0  72.5  36.5  38.8  19.0
 27  599  7200  38.0  12.5  5.4  66.0  34.0  36.5  20.7
 28  551  7950  41.0  13.1  5.3  82.5  37.5  38.4  20.1
 29  592  6100  40.0  13.2  5.5  74.0  33.0  38.5  21.5
 30  642  8500  40.5  13.5  5.0  59.5  30.5  32.4  14.4
 Mean  626  7600  41.0  13.4  5.5  74.9  29.8 36.9   18.6
 S.D.  51  808  1.5  0.6  0.4  9.4  6.0  3.7  2.6

Table 4: Hematological examination/Biochemical examination [control group]

 Number  red blood cell  ehite blood cell  packed cell volume  hemoglobin  serum total protein  GOT GPT   ALP  BUN
   10^4/ mm^3  /mm^3  %  g/dl  g/dl  Karmen  Karmen  King-armstrong  mg/dl
 31  692  5550  40.5  13.7  5.8  53.0  25.0  23.0  16.9
 32  617  10850  41.5  13.5  5.7  72.0  27.0  37.5  21.9
 33  582  9600  43.0  14.2  6.3  57.5  18.0  38.5  18.5
 34  614  6150  41.0  13.7  5.3  59.0  21.5  36.0  18.1
 35  726  7450  44.0  14.4  6.0  65.0  24.5  31.5  20.0
 36  630  7650  39.5  12.8  5.4  64.5  28.0  35.8  21.2
 37  662  9250  40.0  13.1  5.9  69.5  35.5  36.6  15.5
 38  608  6500  40.0  13.2  5.2  69.0  27.0  25.7  18.5
 39  598  8650  40.5  13.1  5.1  60.0  23.0  35.6  28.9
 40  686  6000  39.5 13.4   5.4  57.0  23.5  30.2  14.1
 Mean  642  7765  41.0  13.5  5.6  62.7  25.3  33.0  19.8
 S.D.  45  1680  1.4  0.5  0.4  6.0  4.4  5.0  3.7

Table 5: Absolute organ weight/Relative organ weight [500ppm]

 Number     Liver     Kidney     Spleen     Brain     Testis     Lung     Adrenal gland   Heart  
   g  %  g  %  g  %  g  %  g  %  g  %  g  %  g  %
 1  14.978  4.243  2.819  0.799  0.948  0.269 1.906   0.540  3.148 0.892   1.596  0.452  0.049  0.014  1.145  0.324
 2  13.323  4.298  2.647  0.854  0.831  0.268  1.828  0.590  3.019  0.974  1.360  0.439  0.051  0.016  0.977  0.315
 3  13.635  4.342  2.535  0.807  0.726  0.231  1.891  0.602  3.184  1.014  1.369  0.436  0.052  0.017  1.089  0.347
 4  15.046  4.559  2.673  0.810  1.016  0.308  1.863  0.565  3.182  0.964  1.489  0.451  0.056  0.017  1.248  0.378
 5  15.010 4.276   2.757  0.785  0.755  0.215  1.897  0.540  2.932  0.835  1.622  0.462  0.050  0.014  1.167  0.332
 6  16.275 4.690   2.502  0.721  0.970  0.280  1.833  0.528  2.927  0.844  1.550  0.447  0.051  0.015  1.062  0.306
 7  15.366  4.428  2.820  0.813  0.902  0.260  1.852  0.534  3.221  0.928  1.578  0.455  0.062  0.018  0.991  0.286
 8  16.919  4.674  2.779  0.768  1.154  0.319  1.881  0.520  3.156  0.872  1.706  0.471  0.060  0.017  0.982  0.271
 9  15.783  4.409  2.732 0.763   0.846  0.236  1.861  0.520  3.001  0.838  1.515  0.423  0.051  0.014  1.127  0.315
 10  14.917  4.400  2.874  0.848  0.978  0.288  1.907  0.563  3.226  0.952  1.635  0.482  0.056  0.017  1.044  0.308
Arithmetic Mean  15.125  4.432  2.714  0.797  0.913  0.267  1.872  0.550  3.100  0.911  1.542  0.542 0.054   0.016  1.083  0.318
 S.D  1.029  0.151  0.117  0.038  0.122  0.032  0.027  0.027  0.111  0.061  0.106  0.016  0.004  0.001  0.85  0.029

g: absolute organ weight

%: relative weight

Table 6: Absolute organ weight/Relative organ weight [300ppm]

Number     Liver     Kidney     Spleen     Brain     Testis     Lung     Adrenal gland   Heart  
   g  %  g  %  g  %  g  %  g  %  g  %  g  %  g  %
 11  16.442  4.480  2.719  0.741  0.817  0.226  1.919  0.523  3.095  0.843  1.565  0.426  0.064  0.017  1.072  0.292
 12  13.745  4.140  2.498  0.752  0.794  0.239  1.885  0.568  2.918  0.879  1.565 0.471  0.054  0.016  0.989  0.298
 13  13.042  4.127  2.244  0.710 0.719   0.228  1.999  0.633  3.165  1.002  1.461  0.462  0.041  0.013  0.974  0.308
 14  15.517  4.346  2.739  0.767  1.037  0.290  1.917  0.537  3.059 0.857   1.396  0.391 0.058   0.016  0.997  0.279
 15  14.746  4.262  2.868  0.829  0.792  0.229  1.891  0.547 2.978   0.861  1.407  0.407  0.040  0.012  1.051  0.304
 16  15.906  4.637  2.884  0.841  0.813  0.237  1.907  0.556  3.230  0.942  1.443  0.421  0.048  0.014  1.022  0.298
 17  15.012  4.377  2.975  0.867  0.952  0.278  1.915  0.558  3.450  1.006  1.610  0.469  0.054  0.016  1.073  0.313
18  15.648  4.408  3.105  0.875  0.789  0.222  1.994  0.562  3.192  0.899  1.498  0.422  0.055  0.015  1.145  0.323
 19  15.640  4.258  2.775  0.760  0.916  0.251  1.875  0.514  2.976  0.815  1.621  0.444  0.050  0.014  1.075  0.295
 20  17.861  4.651  2.696  0.702  1.123  0.292  1.892  0.493  3.378  0.880  1.713  0.446  0.055  0.014  1.129  0.294
 Arithmetic Mean  15.346  4.369  2.750  0.784  0.875  0.249  1.919  0.549  3.144  0.898  1.528 0.436   0.052  0.015  1.053  0.300
 S.D  1.278  0.173  0.231  0.060  0.122  0.026  0.041  0.036  0.166  0.062  0.098  0.026  0.007  0.001  0.055  0.012

g: absolute organ weight

%: relative weight

Table 7: Absolute organ weight/Relative organ weight [100ppm]

Number     Liver     Kidney     Spleen     Brain     Testis     Lung     Adrenal gland   Heart  
   g  %  g  %  g  %  g  %  g  %  g  %  g  %  g  %*
 21  15.747  4.845  2.528  0.778  0.984  0.303  1.897  0.584  2.819  0.867  1.513 0.466   0.044  0.014  0.997  0.3
 22  15.326  4.774  2.717  0.846  0.898  0.280  1.890 0.589   2.798  0.872  1.319  0.411  0.043  0.013  0.990  0.3
 23  16.049  4.585 2.751   0.786  0.901  0.257  1.972  0.563  2.857  0.816  1.469  0.420  0.052  0.015  1.148  0.3
 24  14.356  4.044  2.491  0.702  0.904  0.255  1.763  0.479  2.729  0.769  1.446  0.407  0.061  0.017  1.046  0.2
 25  15.000  4.323  2.531  0.729  0.933  0.269  1.869  0.539  3.490  1.006  1.321  0.381  0.060  0.017  1.053  0.3
 26  14.867  4.334  2.679  0.781  0.929  0.271  1.860  0.542  3.218  0.938  1.418  0.413  0.051  0.015  1.128  0.3
 27 15.757   4.414  2.631  0.737  0.768  0.215  1.920  0.538  3.042  0.852  1.549  0.434  0.050  0.014  1.044  0.2
 28  14.777  4.234  2.395  0.686  0.663  0.190  1.859  0.533  2.997  0.859  1.411  0.404  0.052  0.015  1.006  0.2
 29  16.977  4.651  2.525  0.692  0.788  0.216  1.929  0.528  3.429  0.939  1.595  0.437  0.063  0.017  1.025  0.2
 30  13.903  3.972  2.876  0.822  0.921  0.263  1.988  0.568  2.476  0.707  1.578  0.451  0.064  0.018  1.146  0.3
  Arithmetic Mean  15.276 4.418   2.612  0.756  0.869  0.252  1.895  0.546  2.986  0.863  1.462 0.422  0.054   0.016  1.058 0.3 
 S.D  0.846  0.279  0.137  0.052  0.093  0.033  0.061  0.030  0.302  0.082  0.093  0.024  0.007  0.002  0.058  0.01

g: absolute organ weight

%: relative weight

*In the copy of the original study, the last two digits behind the comma could not be identified.
Cause
there are no abnormality findings in the study on the heart, this data gap is negligible and therefore does not lead to a deterioration of the results of this study

Table 8: Absolute organ weight/Relative organ weight [control group]

Number     Liver     Kidney     Spleen     Brain     Testis     Lung     Adrenal gland   Heart  
   g  %  g  %  g  %  g  %  g  %  g  %  g  %  g  %
 31  15.710  4.540  2.412  0.697  0.722  0.209  1.945  0.562  3.194  0.923  1.501  0.434  0.049  0.014  1.043  0.301
 32  16.429  4.861  2.327  0.688  1.102  0.328  1.795  0.531  1.075  0.318  1.656  0.490  0.049  0.014  1.027  0.304
 33  16.141  4.521  2.386  0.668  1.049  0.294  1.840  0.515  3.151  0.883 1.534   0.430  0.063  0.018  1.014  0.284
 34  12.400  3.792  2.092  0.640  0.714  0.218  1.773  0.542  2.127  0.650  1.411  0.431  0.050  0.015  0.798  0.299
 35  15.653  4.397  2.343  0.658  0.889  0.250  1.835  0.515  3.209  0.901  1.479  0.415  0.053  0.015  1.121  0.315
 36  14.892  4.231  2.470  0.702  0.815  0.232  2.005  0.570  3.029  0.861  1.574  0.447  0.057  0.016  1.049  0.298
 37  15.694  4.014  2.762  0.706  0.921 0.236   1.852  0.474  2.662  0.681  1.630 0.417   0.049  0.013  1.061  0.271
 38  12.952  3.669  2.391  0.677  0.800  0.227  1.986  0.563  3.227  0.914  1.479  0.419  0.047  0.013  1.200  0.340
 39  14.818  4.071  2.747  0.755  0.900  0.247  1.922  0.528  3.244  0.891  1.633  0.449  0.046  0.013  1.139  0.313
 40  14.639  4.000  2.449  0.669  1.013  0.277  1.877  0.513  3.329  0.910  1.544  0.422  0.053  0.014  1.068  0.292
Arithmetic Mean  14.933  4.210  2.438  0.686  0.893  0.252  1.883  0.531  2.825  0.793 1.544   0.435 0.052  0.015   1.052  0.302
 S.D  1.259  0.351  0.187  0.030  0.126  0.035  0.075  0.028  0.678  0.184  0.075  0.021  0.005  0.002  0.101  0.018

g: absolute organ weight

%: relative weight

Conclusions:
The male rats were exposed to 500, 300 and 100 ppm 3-methyl-3-methoxybutanol by inhalation for 4 hours a day, 5 days a week (total 20 times of inhalation. Although no severe effect in the animals was noted, the serum transaminase (GOT) activity and absolute and relative kidney weights of the experimental groups were significantly higher than those of control group. As the results, it was considered that liver and kidney were slightly affected by MMB.
Executive summary:

Inhalation exposure to MMB at the concentrations of 500, 300 and 100 ppm for 4 hours a day, 5 days a week (total 20 times of inhalation) produced following findings in male rats.

1) GOT activity in 500 ppm and 100 ppm exposure groups was significantly higher than that of control group.

2) The Absolute and relative kidney weights in all exposure groups were significantly higher than that of control group. However no abnormal findings were noted in liver histopathologically and except for GOT activity, liver function tests showed no abnormalities. Regarding kidney, there was no significant difference between each experimental group and control group in each functional examination and no toxicological relevant change was noted in histopathological examinations.

These results were summarized as follows.

It is considered that under this experiment condition, although MMB exposure slightly affected liver and kidney, MMB exposure caused no severe effect in organs. Therefore, it is necessary to pay attention enough if there is a possibility to inhale MMB for a long time. The chemical’s action to the living body may be different according to the animal species. It would be desirable to examine the influence of this material to the living body further in the greater detail by long term experiment.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
530 mg/m³
Study duration:
subacute
Species:
rat

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.

In a reproduction/developmental toxicity screening test following OECD TG 421 (see section 7.8.1 or CSR section 5.9.1) rats were given MMB by gavage at 0 (vehicle: distilled water), 8, 40, 200 or 1000 mg/kg bw/day. Males were dosed for 47 days and females were dosed from day 14 before mating to day 4 of lactation throughout the mating and pregnancy period. Increases in absolute and relative weights of the kidney in males at 200 mg/kg bw/day and higher and relative weight of the liver and kidney in females at 1000 mg/kg bw/day were detected. No effects of MMB were detected on reprotoxic or developmental parameters. The NOAELs were considered to be 40 mg/kg bw/day in males and 200 mg/kg bw/day in females for general toxicity based on observations of increased kidney weights in both sexes.

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 could be 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. The LOAEC for this repeated dose inhalation toxicity study was considered to be 100 ppm.


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

A 90-day oral study in rats according to OECD 408 was conducted as requested by REACH Annex IX. The purpose was also to assess, if the increase in the relative kidney and liver weight shown in the sub-acute toxicity study (28 -days) and in the reproduction/developmental toxicity screening study is an adverse effect or not. In this study no statistically significant effects were observed concerning organ weight findings including organ / body weight ratios. Thus, the increases in kidney and liver weight can be considered to be not adverse based on this reliable sub-chronic test. The no adverse effect doses derived from the organ weight findings in the old studies have therefore not been further used in the risk assessment.    



Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
In this inhalative study a LOAEC of 530 mg/m^3 air was detected. No NOAEL could be derived. This study was not selected as point of departure for the inhalative DNEL calculation.


Justification for classification or non-classification

The available toxicity data of 3 -methoxy-3 -methylbutan-1 -ol (CAS: 56539 -66 -3) suggests that the substance should not be classified for specific target organ toxicity following repeated oral exposure because there was no evidence of significant toxic effects in a sub-acute toxicity study (28 -days) and a sub-chronic toxicity study (90 -days).