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Description of key information

A combined repeated dose toxicity study with Reproduction/Developmental Toxicity Screening test for 2 -(2 -methylpropxy)ethanol via the oral route was carried out on rats in compliance with GLP and in accordance with the Japanese Guideline "Methods of Testing for new Chemical Substances (2011). Doses of 0, 15, 50 and 150 mg/kg were administered to 12 male and 12 females rats respectively (24 rats in total per dose).The male rats were dissected after 42-days consecutive administration. Test substance was administrated to female rat for 42~47 consecutive days (from two weeks before mating to 4th day of suckle). New-born infant was dissected on the 4th day of suckle while female who delivered a new-born infant was dissected on the 5th. With regard to 0 and 150mg/kg administration, a non-mated females (10/group) were prepared. Half of them are dissected after 42-days consecutive administration while the remaining half and 5 males with dose of 0 and 150 mg/kg were dissected after they had been raised for 14 days.

Administration of test substance did not influence estrus cycle, copulation and pregnancy rate in provided experiment conditions. Pregnancy period, birthrate, delivering rate and suckling state were not influenced by administration of test substance. Administration of test substance did not affect the number of corpus luteum and implantation or implantation rate. Therefore, test substance was considered not to have reproductive toxicity to parental animals in conditions less than 150 mg/kg dose.

Hematologic tests on completion of administration showed a decline of erythrocyte number was observed in both male groups more than 50 mg/kg/day and female groups at 150 mg/kg day. It also revealed decline of haemoglobin concentration in the >50 mg/kg male groups and 150 mg/kg non-mated female group.With regard to bone marrow of thighbone, hyperplasia of erythroblast was observed male group and the female groups which gave birth in conditions more than 15 mg/kg dose, as well as non-mated female group exposed to the 150 mg/kg dose. Increased spleen weightsspleen in 50 mg/kg females who gave birth and 150 mg/kg non-mated females was considered to be caused by the test substance.

The NOEAL was concluded to be 150 mg/kg/day for reproductive and developmental toxicity and <15 mg/kg/day for repeated dose toxicity based on the effects obseved on the blood. A classification of classification as STOT RE 2; H373: May cause damage to organs (blood) through prolonged or repeated exposure (oral route) is recommended in accordance with with Regulation (EC) N° 1272/2008.

The NOEAL was concluded to be 150 mg/kg/day for reproductive and developmental toxicity and <15 mg/kg/day for repeated dose toxicity. A classification of classification as STOT RE 2; H373: May cause damage to organs (blood) through prolonged or repeated exposure (oral route) is recommended in accordance with with Regulation (EC) N° 1272/2008 based on the significant effects on the blood observed at doses >50 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to
Guideline:
other: Japanese guideline “Methods of Testing New Chemical Substances (2011)”
Principles of method if other than guideline:
Test substance was administrated to Crl:CD(SD) rat via oral with 0, 15, 50, and 150 mg/kg test substance (test substance was diluted with distilled water for injection). Each dose was administrated to 12 male and female rats respectively (24 rats in total). The male rats were dissected after 42-days consecutive administration. Test substance was administrated to female rat for 42~47 consecutive days (from two weeks before mating to 4th day of suckle). New-born infant was dissected on the 4th day of suckle while female who delivered a new-born infant was dissected on the 5th day. With regard to 0 and 150 mg/kg administration, non-mated females (10/group) were prepared. Half of them are dissected after 42-days consecutive administration while the remaining half and 5 males with dose of 0 and 150 mg/kg were dissected after they had been raised for 14 days.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
8.5 weeks old Sprague-Dawley [Crl:CD(SD), SPF] purchased from Atsugi Breeding Centre of Charles River Laboratories, Japan
Sex:
male/female
Details on test animals and environmental conditions:
Environmental conditions in rearing room
Temperature:21.0~25.0 °C, humidity: 40.0~75.0%, ventilation: 15/hour, light and darkness cycle 12 hours (7:00~19:00 light, 19:00~7:00 darkness).
Every rat was accommodated in an animal-rearing cage (220w×270d×190h mm) with free access to pellet (CE-2, CLEA Japan, Inc.) and tap water (supply from municipal water department of Hatano city).
Route of administration:
oral: unspecified
Vehicle:
water
Duration of treatment / exposure:
The male rats were dissected after 42-days consecutive administration. Test substance was administrated to female rat for 42~47 consecutive days (from two weeks before mating to 4th day of suckle).
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 male and 12 female
Control animals:
yes, concurrent vehicle
Mortality:
no mortality observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Feed intake of male, mated female and un-mated female groups significantly decreased in the condition of 150 mg/kg dose, resulting in body weight decrease for 4 days. However, influence of test substance on body weight and feed consumption was not significant after 4 days.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A hematologic test was carried out on completion of administration. A decline in erythrocyte number was observed in both male and female groups more than 50 mg/kg dose and non-mated female groups with 150 mg/kg dose. A decline of haemoglobin concentration in male group more than 50 mg/kg dose and non-mated female group with 150 mg/kg dose was also observed. Increase in the ratio of reticulocyte was observed in the male group with 150 mg/kg dose, the female groups which gave birth in conditions more than 50 mg/kg dose, and non-mated female group with 150 mg/kg dose. Histopathological diagnosis of liver and spleen diagnosed extramedullary haematopoiesis. Hyperplasia of erythroblast was observed in bone marrow. Bone-marrow smear test identified decline in the ratio of myelocyte and erythroblast, expansion of red pulp in spleen observed in > 50 mg/kg dose mated female groups as well as in 150 mg/kg dose male and non-mated female groups hematogenous function was increasing in response to chronical anaemia. With regard to bone marrow of thighbone, hyperplasia of erythroblast was observed male group and the female groups which gave birth in conditions more than 15 mg/kg dose, as well as non-mated female group with 150 mg/kg dose. The ration of neutrophilic myelocyte accompanied by increase in erythroblast cell was observed in the female group which gave birth in condition more than 15 mg/kg dose. With regard to extramedullary haematopoiesis of liver observed in one 15 mg/kg dose female, the test substance was considered not to have any relation because there was not change indicating haemolytic anaemia.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
One day after administration, red urine was observed in all 150 mg/kg dose males and females and in the mated 50 mg/kg dose female group. With regard to repeated-dose toxicity test of similar substances such as 2-(1-methylethoxy)ethanol and 2-tert-butoxyethanol via oral adminidtration,red urine was observed one day after administration. Therefore, red urine was considered to be acute effect common in these substances. Urine check conduced on 37th day did not show any influence of test substance except for uric blood in non-mated female. Therefore, the effect of test substance can be most severe when it is administrated initially. Female animals were more susceptible to toxicity than male animals, considering that red urine was identified only in female group exposured to the 50 mg/ kg dose and that paleness and necrosis of the tails were recognised in two females exposed to 150 mg/kg dose.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Measurement of organ weight revealed weight increase of spleen in female groups which gave birth exposed to more than 50 mg/kg dose as well as non-mated female exposed to 150 mg/kg dose. This was considered to be caused by test substance due to changes of increasing extramedullary haematopoiesis. Increase in relative liver weight of female group which gave birth exposed to the 150 mg/kg dose and in absolute and relative liver weight of non-mated female group exposed to 150 mg/ kg dose was more than the ranges of background data, leading to estimate this was accompanied by extramedullary haematopoiesis. With regard to increase in relative heart weight of female group which delivered and in absolute and relative liver weight of non-mated female group, both exposed to 150 mg/kg dose, vicarious hypertrophy in response to anaemia was observed although there was no histopathological change of the myocardium. Increase in relative kidney weight of female group which delivered exposed to 150 mg/kg dose was within the ranges of background data, leading to consider no influence of test substance on weight increase.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological diagnosis of pathologic autopsy at the end of administration observed brown pigmentation increase of spleen, brown pigmentation of Kupper’s liver cell in >50 mg/kg female groups which gave birth as well as 150 mg/kg dose non-mated females. Furthermore, brown pigmentation of kidney’s proximal tubule was considered to be hemosiderin pigmentation originated from haemolyzed haemoglobin, which was possibly due to haemolytic anaemia induced by test substance. With regard to changes of tips colour into black in 150 mg/kg dose female group which gave birth, necrosis and ulcer were observed. This indicated circulatory disturbance due to haemolytic anaemia caused by haemolysis. Similar substance, 2-butoxyethanol decrease ATP in erythrocyte and cause haemolysis due to its metabolite, 2-butoxyacetate. Test substance was also considered to have caused haemolysis for a similar mechanism. Although 2-butoxyethanol is also reported to induce testicular atrophy, findings indicating testicular toxicity were not identified based on testis weight and histopathological diagnosis of testis conducted in this report.
Other effects:
no effects observed
Description (incidence and severity):
Reproductive and developmental toxicity
Administration of test substance did not influence estrus cycle, copulation and pregnancy rate in provided experiment conditions. Pregnancy period, birthrate, delivering rate and suckling state were not influenced by administration of test substance. Administration of test substance did not affect the number of corpus luteum and implantation or implantation rate. Therefore, test substance was considered not to have reproductive toxicity to parental animals exposed to less than 150 mg/kg dose. Administration of test substance did not affect survival rate, morphology and weight (0 and 4th day of suckling) of new-born, which lead to conclude that less than 150 mg/kg administration of test substance did not influence next generation in conditions used in this report.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive and Developmental toxicity
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
blood
erythrocyte development
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
No effects on reproduction were observed during this study and the NOEAL for reproductive effects was therefore considered to be 150 mg/kg/day, this being the highest dose tested. Based on the results of the study the substance should not be classified as toxic to reproduction in accordance with Regulation (EC) N° 1272/2008.

RBC was significantly decreased at 50 mg/kg bw/d (p<0.01) and 150 mg/kg bw/d (p<0.01), and other blood parameters were significantly different from the control at 150 mg/kg bw/d (p<0.01) in males at the end of the treatment period. In females RBC significantly decreased at 150 mg/kg bw/d (p<0.01). Taking into account these effects, classification as STOT RE 2; H373: May cause damage to organs (blood) through prolonged or repeated exposure (oral route) is recommended inaccordance with with Regulation (EC) N° 1272/2008.
Executive summary:

A combined repeated dose toxicity study with Reproduction/Developmental Toxicity Screening test for 2 -(2 -methylpropxy)ethanol via the oral route was carried out on rats in compliance with GLP and in accordance with the Japanese Guideline "Methods of Testing for new Chemical Substances (2011). Doses of 0, 15, 50 and 150 mg/kg were administered to 12 male and 12 females rats respectively (24 rats in total per dose).The male rats were dissected after 42-days consecutive administration. Test substance was administrated to female rat for 42~47 consecutive days (from two weeks before mating to 4th day of suckle). New-born infant was dissected on the 4th day of suckle while female who delivered a new-born infant was dissected on the 5th. With regard to 0 and 150mg/kg administration, a non-mated females (10/group) were prepared. Half of them are dissected after 42-days consecutive administration while the remaining half and 5 males with dose of 0 and 150 mg/kg were dissected after they had been raised for 14 days.

Administration of test substance did not influence estrus cycle, copulation and pregnancy rate in provided experiment conditions. Pregnancy period, birthrate, delivering rate and suckling state were not influenced by administration of test substance. Administration of test substance did not affect the number of corpus luteum and implantation or implantation rate. Therefore, test substance was considered not to have reproductive toxicity to parental animals in conditions less than 150 mg/kg dose.

One day after administration, red urine was observed in all the males and females in the 150 mg/kg dose group and in the mated 50 mg/kg female group with dose and was considered an acute affect of the substance.Urine check conducted on 37th day did not show any influence of test substance except for uric blood in non-mated female. Therefore, the effect of test substance can be most severe when it is administrated initially.Hematologic tests on completion of administration showed a decline of erythrocyte number was observed in >50 mg/kg males and 150 mg/kg female groups. It also revealed a decline of haemoglobin concentration in the > 50 mg/kg male groups and 150 mg/kg non-mated female group.With regard to bone marrow of thighbone, hyperplasia of erythroblast was observed male group and the female groups which gave birth exposed to more than 15 mg/kg dose, as well as non-mated female 150 mg/kg dose group. Increased spleen weightsspleen in 50 mg/kg females who gave birth and 150 mg/kg non-mated females was considered to be caused by the test substance.

The NOEAL was concluded to be 150 mg/kg/day for reproductive and developmental toxicity and <15 mg/kg/day for repeated dose toxicity. A classification of classification as STOT RE 2; H373: May cause damage to organs (blood) through prolonged or repeated exposure (oral route) is recommended in accordance with with Regulation (EC) N° 1272/2008 based on the significant effects on the blood observed at doses >50 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
haematopoietic
Organ:
blood
erythrocyte development

Additional information

Justification for classification or non-classification

A combined repeated dose toxicity study with Reproduction/Developmental Toxicity Screening test for

2 -(2 -methylpropxy)ethanol via the oral route was carried out on rats. The Haematological observations found

RBC was significantly decreased at 50 mg/kg bw/d (p<0.01) and 150 mg/kg bw/d (p<0.01) and other blood parameters were significantly different from the control at 150 mg/kg bw/d (p<0.01) in males at the end of the treatment period. In females significant effects at the end of the recovery period at 150 mg/kg bw/d (p<0.01).

Taking into account the values reported in Table 3.9.3 of the CLP Regulation from a classification as STOT RE, adapted for a 28-day repeated-dose toxicity study via the oral route, a classification as STOT RE 2; H373: May cause damage to organs (blood) through prolonged or repeated exposure (oral route) should apply.