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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.

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Administrative data

Description of key information

The Category member 1, Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid, was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days in an oral subacute rat study according to OECD guideline 407. On the basis of this 28-Day Repeated Dose Oral Toxicity study with Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid in male and female Wistar rats with dose levels of 50, 250, and 1000 mg/kg body weight per day revealed no major toxicity or mortality. No mortality or signs of toxicity of Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid were found up to a dose level of 1000 mg/kg bw/day. Therefore, the NOAEL of the test item in this study is considered to be 1000 mg/kg bw/day.

An oral study according to OECD 422 was done on the Source Substance 2, docosanoic acid. The NOAEL of the study was 1000 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
On the basis of all evaluated data, the similarity of all category members of the ISOCARB is justified on basis of the physico-chemical properties, toxicological and ecotoxicological profiles. There is convincing evidence that these chemicals possess an overall common category profile.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Hino, Tokyo
- Age at study initiation: 8 weeks
- Weight at study initiation: male: 312.1 - 363.7 g; female: 205.3 - 230.8 g
- Housing: metal wire floor cages
- Diet: ad libitum CE-2, CLEA Japan
- Water: ad libitum tap water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1
- Humidity (%): 50 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
prepared more frequently than once a week; aliquots were kept by each concentration refrigerated in airtight conditions

VEHICLE
- Justification for use and choice of vehicle (if other than water): due to insolubility in water
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): V6H2050
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
- males: 42 days
- females: from 14 days prior to mating to day 3 of lactation
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/d
Basis:
other: nominal conc.
No. of animals per sex per dose:
13 in each group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on preliminary result in a 14 day-repeated dose toxicity study, where no signs of toxicity were found
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day

BODY WEIGHT: Yes
- Time schedule for examinations: males: days 1, 8, 15, 22, 29, 36, 42; pregnancy females: premating days 1, 8, 15; pregnancy days: 0, 7, 14, 20; lactation days: 0, 4; non pregnancy females: day 1, 8, 15, 22, 25

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (males only)
- Time schedule for collection of blood: prior to autopsy
- Anaesthetic used for blood collection: Yes (identity): pentobarbital sodium
- Animals fasted: 18 - 24 hours before sacrifice
- How many animals: all surviving animals
- Parameters checked: red blood cell count (RBC), white blood cell count (WBC), haemoglobin content (Hb), mean corpuscular volume (MCV), haematocrit (Ht), mean corpuscular haemoglobin content (MCH), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes (males only)
- Animals fasted: 18 - 24 hours before sacrifice
- How many animals: all surviving animals
- Parameters checked: total protein, albumin, total cholesterol concentration, glucose, urea nitrogen, creatinine, alkaline phosphatase activity, GOT, GPT, γ-GTP, triglyceride concentration, inorganic phosphorus, total bilirubin, calcium, sodium, potassium, chlorine, A/G ratio

URINALYSIS: No

Sacrifice and pathology:
Organ weights:
male: heart, liver, kidneys, thymus, testes, epididymides
female: heart, liver, kidneys, thymus

Pathological findings and histopathology (control and 1000 mg/kg bw /day group):
male: heart, liver, spleen, kidney, adrenal, testis, epididymis, brain, thymus, bladder
female: brain, liver, spleen, thymus, kidney, adrenal, bladder, heart, ovary
Statistics:
Yates test, Mann-Whitney U test, Fisher exact test, Bartlett test, Dunnett test, Scheffe test, Kruskal-Wallis test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
significant increase was observed in 100 mg/kg bw/day group (non-adverse)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
significant decrease was observed in 100 mg/kg bw/day group during lactation (non-adverse)
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):
significant decrease of mean corpuscular haemoglobin concentration in the 300 and 1000 mg/kg bw/day groups (non-adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
non-adverse
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
non-adverse
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
non-adverse
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
non-adverse
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- Males: No deaths or abnormalities in general condition were observed in any of the treated groups
- Females: No deaths were observed in any treated group

BODY WEIGHT AND WEIGHT GAIN
- Males: significant increase (p<0.01) compared to control was observed in 100 mg/kg bw/day group between 8 - 15 days and between 15 - 29 days. However, since no change was observed in other groups, it was considered not related to the dosing of compound.
- Females: no significant change in body weight was noted

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Males: no changes in food consumption related to the dosing of compound
- Females: significant decrease (p<0.01) compared to control was observed during lactation in 100 mg/kg bw/day group. However, since no other changes in food consumption were noted in 300 mg/kg bw/day and 1000 mg/kg bw/day, it was not related to the dosing with the compound.

HAEMATOLOGY
- Males: a significant decrease (p<0.01) of mean corpuscular haemoglobin concentration (MCHC) compared to control was found in the 300 and 1000 mg/kg bw/day dose group, respectively. No other differences were noted.

CLINICAL CHEMISTRY
- Males: a significant decreased ALP level (p<0.05) compared to control was found in all dosing groups. A significant decreased glucose level (p<0.05) compared to control was found in the high dose group. While a significant increase in chloride (p<0.05) was found in the 300 mg/kg bw/day group, a significant decrease in calcium content (p<0.01) and in total protein (p<0.05) was noted in this group. However, all the findings lack a dose-dependency and were regarded as incidental.

ORGAN WEIGHTS
- Males: in 100 mg/kg bw/day males, the actual weight ratio of liver weight compared to control values were increased (p <0.05) . No other significant differences were found in all groups. Thus, this finding lacks a dose-dependency and was regarded as incidental.
- Females: actual kidney weight was significantly reduced (p<0.05) in the 100 mg/kg bw/day group. No other weight changes were noted. Thus, this finding lacks a dose-dependency and was regarded as incidental.

GROSS PATHOLOGY
Males:
- Heart: myocardial degeneration was noted in one animal of the control group and the 1000 mg/kg bw/day group, respectively
- Liver: periportal fatty change of the liver was found in all animals of the control and the high dose group. Focal necrosis was noted in one animal of the high-dose group.
- Spleen: all animals of the control and the high-dose group showed brown pigment deposits of the spleen as well as extramedullary hematopeiosis
- Kidney: while fibrosis was only found in 1/13 animals of the control group, eosinophilic bodies and basophilic tubules in the cortex were noted in animals of both control and 1000 mg/kg bw/day groups
- Adrenal gland: one animal of the high-dose group showed fibrosis
- Testis: atrophy of the seminiferous tubules was observed in two animals of the 1000 mg/kg bw/day dose group, with one animal affected on both sides and one side affected in the other animal. No other abnormalities were noted.
- Epididymis: abnormal sperm granuloma was found in one animal of the control group
- Brain: no abnormal findings noted
- Thymus: no abnormal findings noted
- Bladder: no abnormal findings noted

Females:
- Brain: one animal of the high-dose group showed abnormal mineral deposition in the thalamus
- Liver: focal necrosis and fibrosis was found in one animal of the control group, while one animal of the high-dose group showed only focal necrosis
- Spleen: brown pigmentation and extramedullary hematopeiosis was found in animals of the control and the high-dose group, but no varying degrees of frequency was observed
- Thymus: atrophy and bleeding was observed in animals of both groups, control and high-dose group, respectively
- Kidney: basophilic tubules of the cortex and a dilatation of the renal pelvis was seen in the control and high-dose group
- Adrenal gland: cortical necrosis was observed in one animal of the control group
- Heart: no abnormal findings noted
- Bladder: no abnormal findings noted

Key result
Dose descriptor:
NOAEL
Remarks:
repeated dose toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse substance-related systemic effects were noted
Key result
Critical effects observed:
not specified
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
On the basis of all evaluated data, the similarity of all category members of the ISOCARB is justified on basis of the physico-chemical properties, toxicological and ecotoxicological profiles. There is convincing evidence that these chemicals possess an overall common category profile (Please see Category approach).
Reason / purpose for cross-reference:
read-across source
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred in the control or any of the dose groups during the treatment or recovery period of this study.
During the treatment period, slight to severe salivation was noted occasionally in some males and females of the main study and recovery HD group. Furthermore, moving the bedding was observed in all males and all females of the main study and recovery HD group during the treatment days 6-28 in males and 5-28 in females. As the symptoms of salivation and moving the bedding were noted mainly immediately after administration and just for a short period, these signs were considered to be a sign of discomfort due to a local reaction to the test item rather than a systemic adverse effect.
Low incidences of clinical signs like slight to moderate piloerection and slightly increased spontaneous activity were noted in isolated males and/or females of the LD and HD group main study and recovery animals during the treatment period. As these slight clinical signs were mostly transient and seen irrespective of the groups in isolated animals, they were considered to be incidental. During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant effects of test item on body weight and body weight gain were observed in the LD and the MD groups of both males and females during treatment and recovery period. However, overall body weight gain (day 1-28) in male main study animals was observed to be statistically significantly lower in MD and HD group when compared with the controls. In females, mean body weight was marginally higher in the HD group compared to the control group at the end of the treatment period. However, body weight was comparable during the recovery period without achieving statistical significance. Overall mean weight gain (Day 1-28) during the treatment period was slightly and statistically insignificantly higher in the main study female HD group when compared to the controls. During the recovery period, mean daily body weight gain was slightly reduced in the HD group compared to the control group.
As differences were marginal, decrease observed in males and values were within the normal range of variation for animals of this strain and in the absence of major clinical signs and good overall health of the animals, effects on body weight were not considered as an adverse effect of the test item.

FOOD CONSUMPTION
There were no statistically significant effects on food consumption during the treatment period and recovery period in both males and females of the dose groups when compared to the respective controls except statistically significantly lower average daily food consumption was observed in HD recovery females. As no such effect was observed during treatment period, this significant effect during recovery period was considered to be incidental and not related to the treatment. During the treatment period, overall group mean food consumption (Day 1-28) of the main study male was slightly lower when compared with the respective control which was in correlation with respective body weights.

OPHTHALMOSCOPIC EXAMINATION
No ophthalmologic findings were observed in any of the animals of this study.

HAEMATOLOGY
In males sacrificed at the end of treatment period, statistical analysis revealed statistically significantly lower mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), platelet count (PLT), white blood cells (WBC) and higher monocytes values in HD group when compared with the controls. In females sacrificed at the end of treatment period, there was a marginally but statistically significantly lower mean value of mean corpuscular haemoglobin (MCH) observed in the MD and the HD group when compared to the control group. As the respective values were marginally higher or lower and within the normal range of variation for animals of this strain and age, differences are not assumed to be biologically relevant although statistically significant.
At the end of the recovery period, with exception of reticulocytes and platelet count (PLT), no statistically significant difference was observed in males in any of the haematology parameters between the dose groups and the control group. Marginally but statistically significantly higher reticulocytes and platelet count (PLT) values in the male HD group were within the normal range of variation and were not assumed to be biologically relevant.
Statistically significant and marginally lower MCH and MCHC in the female HD group observed at the end of the recovery period was considered incidental with values being in the normal range of variation.
Besides, all haematological parameters and blood coagulation parameters in males and females were in the normal range of variation and no test item-related effects were observed.

CLINICAL CHEMISTRY
In male animals, at the end of the treatment period, alanine aminotransferase (ALAT), total protein (TP) in HD and cholesterol in MD and HD were observed to be marginally but statistically significantly lower when compared to the control group. As all values were within the normal range of variation, this effect was considered to be incidental. In female animals, total protein (TP) and Albumin (Alb) were marginally but statistically significantly lower in the HD group at the end of the treatment period compared to the control group. This effect is not assumed to be biologically relevant, as the difference is marginal and values were within the normal range of variation. In males and females, the observed significant effect on parameters of clinical biochemistry did not persist up to the end of recovery and were not considered to be adverse.

URINALYSIS
Slightly higher leukocyte levels were found in the urine of one male animal of HD recovery group and two animals of control recovery group. No such trend was observed in the female animals and main study males sacrificed at the end of treatment period. In males and females, all other urinary parameters were in the normal range of variation, and no test item-related differences between the dose groups and control group were observed.

NEUROBEHAVIOUR
In males and females, no relevant effects were observed in any of the parameters of the functional observation battery at the end of the treatment period or recovery period.

ORGAN WEIGHTS
At the end of the treatment period, absolute adrenals, thymus, thyroid/parathyroid weights in HD and absolute epididymides weights in LD and HD were statistically significantly lower in male animals than in the respective controls. Absolute spleen weights in MD group were also statistically significantly lower when compared to the control group. The relative (to brain weight) liver and kidney weights in LD and MD, spleen weights in MD, thymus weights in MD and HD, epididymides weights in LD,MD and HD and thyroid/parathyroid weights in LD and HD were statistically significantly lower when compared to the control group. A statistically significantly higher relative (to body weight) brain and testes weights in all dose groups and statistically significantly lower relative (to body weight) thymus and thyroid/parathyroid weights in HD group were observed when compared with the controls. In female animals sacrificed at the end of treatment period, a statistically significantly higher absolute and relative (to body and brain weight) liver weight was found in the female HD group when compared to the controls. There was statistically significantly lower absolute and relative (to body weight) thymus weights observed in HD group when compared with the controls. A statistically significantly lower absolute and relative (to brain and body weight) thyroid/parathyroid weights in LD and statistically significantly higher ovary weights relative to body weights were observed in HD group when compared with the controls. At the end of the recovery period, there were no statistically or biologically significant effects on the absolute and relative organ weights in the male and female dose groups except statistically significantly higher relative (to brain and body weight) ovary weights in HD group females when compared to the respective controls. As no such effect was observed in main study females, this effect on recovery female ovary weight was considered to be incidental and not related to the treatment with the test item. To these achieved statistical significances, no histological correlate was found for the increased or decreased weights. In the light of absence of adverse histopathological findings in the organs up to HD group and observed histopathological lesions recovered after a 14-day withdrawal period and as this increase or decrease in organ weight was minimal, it was not considered to be adverse. At the end of the treatment period and the recovery period, values for the remaining male and female organs from the dose groups were comparable with the control group.

GROSS PATHOLOGY
Predominant macroscopic findings observed in male animals sacrificed at the end of treatment period were yellow spot on right epididymides and large sized mandibular lymphnodes in 1 each male of the HD group. In the male animals sacrificed at the end of recovery period, the yellow spots on epididymides in 2/5 males and discolored dark ileum /payer’s patches were observed in 1/5 males of the control recovery group. In female animals sacrificed at the end of treatment period, dilated right renal pelvis in 1/5 females of the control group and fluid distension in the uterus of 2/5 HD group females was observed. No macroscopic findings observed at necropsy in any of the female animals sacrificed at the end of recovery period. These gross pathological findings were spontaneous in nature and as such not considered to be a systemic effect due to test item administration. Histopathologically also these gross lesions could not be related to treatment with the test item. Spots on epididymides correlated microscopically with the spermatid granulomas of epididymis, the congestion in the ileum, the pelvic dilation of the kidney and the cornual dilation of the uterus related to estrus cyclic changes, respectively. These findings are common in rats and were within the range of normal background lesions which may be recorded in animals of this strain and age and therefore considered not to be treatment-related. Microscopically, enlarged (large sized) mandibular lymph node in HD group male sacrificed at the end of treatment period was a poorly differentiated salivary gland carcinoma. This type of carcinoma occasionally occur in rats, even in the young generation and the neoplasm recorded in this study was deemed to be of a spontaneous in nature.

HISTOPATHOLOGY: NON-NEOPLASTIC
Under the conditions of this study, the microscopic findings which were considered to be attributable to treatment with the test item were recorded in the liver and thymus of both sexes. Hepatocellular hypertrophy was recorded in both sexes of the non-recovery group 4 (1000 mg/kg bw/day). This was characterized mainly by centrilobular hypertrophy, and in some locations of the female liver it looked diffusely hypertrophic. There were neither further indicator of cellular injuries such as necrosis or apoptosis nor indicator of cellular proliferation such as increased mitotic figures or polyploidy, in any animals examined under the condition of this study. Therefore, this finding was considered to be of adaptive character and not to be adverse under the condition of this study. Hepatocellular hypertrophy was no longer present in any recovery animals (see table 1 below).
Increased incidence and/or severity of thymic atrophy/involution were recorded in both sexes of the non-recovery group 4 (1000 mg/kg bw/day). No abnormal histological findings were observed in the other lymphoid organs and tissues including spleen and lymph nodes of animals examined in this study, and therefore, it is unlikely that this was attributed to treatment with the test-item. Rather, it is likely to be a secondary response to the stressful condition due to high-dose exposure of the test item, and it was considered not to be of adverse character. In the recovery animals, there were no differences in incidence and severity between the control and the high-dose groups (see table 2 below).
As a result of microscopic examination of testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus with cervix and vagina, no treatment-related effects were observed in any animals examined in this study.
The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.
In the recovery animals, there were no differences in incidence and severity between the control and the high-dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect
Key result
Critical effects observed:
not specified
Conclusions:
On the basis of this 28-Day Repeated Dose Oral Toxicity study with the Category Member 1, Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid, in male and female Wistar rats with dose levels of 50, 250, and 1000 mg/kg body weight day revealed no major toxicity or mortality. No mortality or signs of Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid were found at the dose level of 1000 mg/kg bw/day. Therefore, the NOAEL of the test item in this study is considered to be 1000 mg/kg bw/day.
Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of the Category Member 1, Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid, via oral administration to rats over a period of 28 days.

The Category member 1 was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. The 4 groups comprised 5 male and 5 female Wistar rats each. To detect possible delayed occurrence or persistence of, or recovery from toxic effects, animals in the recovery group were observed for a period of 14 days following the last administration. The following doses were evaluated: control (0 mg/kg bw), low dose (50 mg/kg bw), medium dose (250 mg/kg bw), high dose (1000 mg/kg bw). The test item formulation was prepared freshly on each day of administration. The test item was suspended in corn oil and administered daily during a 28-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements. During the period of administration, the animals were observed precisely each day for signs of toxicity. Body weight and food consumption were measured weekly. Once before the first exposure and once in the last week of exposure as well as in the last week of the recovery period, multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests. Haematological, blood coagulation and clinical biochemistry examinations were made on blood samples obtained from the overnight fasted animals at the terminal sacrifice. A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. At the conclusion of the test, all animals were sacrificed and observed macroscopically. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. A full histopathological evaluation of the tissues was performed on high dose and control animals. These examinations were extended to liver, thymus and stomach of animals of all other dosage groups and recovery group as treatment-related changes were observed. All gross lesions macroscopically identified were examined microscopically in all animals. Formulation samples were taken at various intervals for analysis of nominal concentrations, stability and homogeneity and stored between -15 to -35 °C until analysis at the end of the study.

No mortality occurred in the control or any of the dose groups during the treatment and recovery period of this study.

During the treatment period, slight to severe salivation was noted occasionally in some males and females of the main study and recovery HD group. Furthermore, moving the bedding was observed in all males and all females of the main study and recovery HD group during the treatment days 6-28 in males and 5-28 in females. As the symptoms of salivation and moving the bedding were noted mainly immediately after administration and just for a short period, these signs were considered to be a sign of discomfort due to a local reaction to the test item rather than a systemic adverse effect.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

No ophthalmologic findings were observed in any of the animals of this study.

In males and females, no relevant effects were observed in any of the parameters of the functional observation battery at the end of the treatment period or recovery period. There were no biologically relevant differences in body temperature between the groups.

No statistically significant effects of test item on body weight and body weight gain were observed in the LD and the MD groups of both males and females during treatment and recovery period. However, overall body weight gain (day 1-28) in male main study animals was observed to be statistically significantly lower in MD and HD group when compared with the controls. In females, mean body weight was marginally higher in the HD group compared to the control group at the end of the treatment period. However, body weight was comparable during the recovery period without achieving statistical significance. Overall mean weight gain (Day 1-28) during the treatment period was slightly and statistically insignificantly higher in the main study female HD group when compared to the controls. During the recovery period, mean daily body weight gain was slightly reduced in the HD group compared to the control group. As differences were marginal, decrease observed in males and values were within the normal range of variation for animals of this strain and in the absence of major clinical signs and good overall health of the animals, effects on body weight were not considered as an adverse effect of the test item.

There were no statistically significant effects on food consumption during the treatment period and recovery period in both males and females of the dose groups when compared to the respective controls except statistically significantly lower average daily food consumption was observed in HD recovery females. As no such effect was observed during treatment period, this significant effect during recovery period was considered to be incidental and not related to the treatment.

During the treatment period, overall group mean food consumption (Day 1-28) of the main study male was slightly lower and in females it was slightly higher when compared with the respective controls which was in correlation with respective body weights.

In males and females sacrificed at the end of treatment period and recovery period, no test item-related effect of toxicological relevance was observed for haematology and coagulation parameters. Values for few parameters were marginally higher or lower but within the normal range of variation for animals of this strain and age. These differences are not assumed to be biologically relevant although statistically significant.

In male animals, at the end of the treatment period, alanine aminotransferase (ALAT), total protein (TP) in HD and cholesterol in MD and HD were observed to be marginally but statistically significantly lower when compared to the control group. As all values were within the normal range of variation, this effect was considered to be incidental. In female animals, total protein (TP) and Albumin (Alb) were marginally but statistically significantly lower in the HD group at the end of the treatment period compared to the control group. This effect is not assumed to be biologically relevant, as the difference is marginal and values were within the normal range of variation.

At the end of the recovery period, blood biochemistry values of the male and female HD group were within the normal range of variation for this strain and comparable to the respective controls. Marginally and statistically significantly lower cholesterol in the male recovery HD group was considered to be incidental.

Slightly higher leukocyte levels were found in the urine of one male animal of HD group and two control group animals at the end of the recovery period. No such trend was observed in the female animals and main study males sacrificed at the end of treatment period.

At necropsy, few gross pathological findings were observed and those were considered to be spontaneous in nature and as such not related to the systemic effect of the test item. Histopathologically, also these gross lesions could not be related to treatment with the test item. The microscopic findings which were considered to be attributable to treatment with the test item, were recorded in the liver and thymus of both sexes.

Hepatocellular hypertrophy was recorded in both sexes of the non-recovery group 4 (1000 mg/kg bw/day). This was characterized mainly by centrilobular hypertrophy, and in some locations of the female liver it looked diffusely hypertrophic. There were neither further indicator of cellular injuries such as necrosis or apoptosis nor indicator of cellular proliferation such as increased mitotic figures or polyploidy, in any animals examined. Therefore, this finding was considered to be of adaptive character and not to be adverse. Hepatocellular hypertrophy was no longer present in any recovery animals. Increased incidence and/or severity of thymic atrophy/involution were recorded in both sexes of the non-recovery group 4 (1000 mg/kg bw/day). No abnormal histological findings were observed in the other lymphoid organs and tissues including spleen and lymph nodes of animals examined in this study, and therefore, it is unlikely that this was attributed to treatment with the test-item. Rather, it is likely to be a secondary response to the stressful condition due to high-dose exposure of the test item, and it was considered not to be of adverse character. In the recovery animals, there were no differences in incidence and severity between the control and the high-dose groups. As a result of microscopic examination of testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus with cervix and vagina, no treatment-related effects were observed in any animals examined in this study. The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Statistical analysis of organ weight data from male and female animals sacrificed at the end of treatment or recovery revealed few significant increases or decreases. However, to these achieved statistical significance, no histological correlate was found for the increased or decreased weights. In the light of absence of adverse histopathological findings in the organs up to HD group and observed histopathological lesions recovered after a 14-day withdrawal period and as this increase or decrease in organ weight was minimal, it was not considered to be adverse.

Dose formulation analysis for nominal concentration, stability and homogeneity revealed that all dose formulations were stable and homogenous at the time of application throughout the study period.

On the basis of this 28-Day Repeated Dose Oral Toxicity study with Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid in male and female Wistar rats with dose levels of 50, 250, and 1000 mg/kg body weight per day revealed no major toxicity or mortality. No mortality or signs of toxicity of Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid were found up to a dose level of 1000 mg/kg bw/day. Therefore, the NOAEL of the test item in this study is considered to be 1000 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 2

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

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

Justification for classification or non-classification

Based on the test results of the Isocarb Category 2 -butyloctanoic acid does not need to be classified according to the Regulation (EC) No 1272/2008.