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Description of key information

A reliable repeated dose (28 day) oral study in rats reports an NOAEL of 50 mg/kg bw/d and a LOAEL of 200 mg/kg bw/d for decreased motor activity, liver effects, alterations in clinical chemistry and urinalysis parameters. 
In a 90 d oral toxicity study (OECD 408), peroxisomal proliferation was detected in high-dose males and females and mid-dose females and alpha-2u in mid and high dose males; no effects have been noted at the lowest dose; therefore, the NOAEL of this study was set at 5 mg/kg/d

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Age at study initiation: 42 +/- 1 day
- Weight at study initiation: 154 +/1 2g
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): Kliba maintenance diet mouse/rat “GLP”, meal ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
i-Nonanoic acid was applied as an emulsion. To prepare this emulsion, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced weekly. The
administration volume was 4 mL/kg body weight.
The stability of the test substance in corn oil for a period of 7 days at room temperature was proven before the start of administration period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance preparations were carried out as a separate study at the Competence Center Analytics, Department of BASF SE, Ludwigshafen, Germany
Duration of treatment / exposure:
92 d (males), 93 d (females)
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
120 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males and females
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were distributed according to weight among the individual test groups, separated by gender. The weight variation of the animals used did not exceed 20 percent of the mean weight of each gender.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals

BODY WEIGHT: Yes
- Time schedule for examinations: At day 0, then weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Determined weekly over a period of 1 day and calculated as mean food consumption grams per animal and day.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly over a period of 4 days and calculated as mean water consumption grams per animal and day

OPHTHALMOSCOPIC EXAMINATION: Yes
- Prior to the start of administration period, eyes of all animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after treatment with a mydriatic agent. At the end of the administration period, eyes of the control and highest dose group animals were re-examined on study day 91.

HAEMATOLOGY: Yes


CLINICAL CHEMISTRY: Yes / No / No data
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters

URINALYSIS: Yes / No / No data
The dry chemical reactions on test strips (Combur-10-test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany).

NEUROBEHAVIOURAL EXAMINATION: No



Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:
Cyaninde-insensitive Palmitoyl-CoA-oxidation in liver homogenate was measured with an automatic analyzer.
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals and sperm motility examined.
Statistics:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed in 9 male and all female animals of test group 3 (120 mg/kg bw/d) shortly after
treatment (<2 hours) on several days of the study starting on study day 12.
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:
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
In females of test group 3 (120 mg/kg bw/d) albumin levels were decreased. In males of test group 3 (
120 mg/kg bw/d) total bilirubin and triglyceride values were decreased.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
significantly increased (15% and 17%, respectively). The relative kidney weight of male animals in test group
2 (30 mg/kg bw/d) was also increased (8%). Furthermore, liver weights were increased in males and fem
ales in the mid and high dose groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The kidneys of two males of test group 3 (120 mg/kg bw/d) showed a light brown discoloration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treated males revealed a dose-response related increase in eosinophilic droplets which were diagnosed
using Mallory Heidenhain-stained slides. The eosinophilic droplets were proven to be alpha 2u globulin
by immunohistochemistry. As secondary effects to the alpha 2u globulin storage in the kidney granular
casts and basophilic tubules were observed in males of test group 2 and 3 (30 and 120 mg/kg bw/d). G
ranular casts are characterized by marked dilation of solitary tubules with lightly staining, granular eosin
ophilic debris, which is derived from exfoliated cortical cells engorged with protein. The basophilic tubules
were regarded to be an attempt of the kidney to regenerate. Females of test groups 2 and 3 (30 and 120
mg/kg bw/d) revealed a minimal increase of vacuolation in the kidney that was proven to be fat by OROstained
slides.
Males of test group 3 (120 mg/kg bw/d) and females of test groups 2 and 3 (30 and 120 mg/kg bw/d)
revealed a minimal to slight vacuolation of hepatocytes in the periportal area (zone 1). These vacuoles
stained positive with ORO-stain and, therefore, were proven to be fat vacuoles.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):

Estrous cycle:
No test substance-related effects on estrous cycle length and the number of cycles were obtained.

Sperm parameters:
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as w
ell as the sperm head counts in the testis and in the cauda epididymidis treatment-related effects were not
observed.

PAL CoA in liver homogenate:
In male and female rats of test group 3 (120 mg/kg bw/d) and additionally in females of test group 2 (30
mg/kg bw/d), cyanide-insensitive Palmitoyl-CoA oxidation in liver tissue was increased. Increases in
Palmitoyl-CoA oxidation are a sign for peroxisomal proliferation
Details on results:
CLINICAL SIGNS AND MORTALITY
No animal died prematurely in the present study.
Salivation was observed in 9 male and all female animals of test group 3 (120 mg/kg bw/d) shortly after treatment (<2 hours) on several days of the study starting on study day 12.

BODY WEIGHT AND WEIGHT GAIN
No test substance-related adverse changes in body weight or body weight gain were observed for male and female animal of test groups 1-3 (5, 30 and 120 mg/kg bw/d) when compared to control groups.

FOOD AND WATER CONSUMPTION
No test substance-related changes in food and water consumption were observed.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related effects were observed.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
In females of test group 3 (120 mg/kg bw/d) albumin levels were decreased. In males of test group 3 (120 mg/kg bw/d) total bilirubin and triglyceride values were decreased. Bilirubin values were below the historical control range, but there was no indication of an anemia among these individuals and, therefore, it was most probable that an increased conjugation rate of bilirubin led to an increased excretion via bile. This effect was regarded as adaptive and not adverse. Triglyceride values in males of test group 3 (120 mg/kg bw/d) were within the historical control range and, therefore, their change was regarded as incidental and not treatment-related.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.

ORGAN WEIGHTS
The absolute and relative kidney weight of male animals in test group 3 (120 mg/kg bw/d) was significantly increased (15% and 17%, respectively). The relative kidney weight of male animals in test group 2 (30 mg/kg bw/d) was also increased (8%). Furthermore, liver weights were increased in males and females in the mid and high dose groups.

GROSS PATHOLOGY
The kidneys of two males of test group 3 (120 mg/kg bw/d) showed a light brown discoloration. This finding was regarded to be treatment-related.
All other findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treated males revealed a dose-response related increase in eosinophilic droplets which were diagnosed using Mallory Heidenhain-stained slides. The eosinophilic droplets were proven to be alpha 2u globulin by immunohistochemistry. As secondary effects to the alpha 2u globulin storage in the kidney granular casts and basophilic tubules were observed in males of test group 2 and 3 (30 and 120 mg/kg bw/d). Granular casts are characterized by marked dilation of solitary tubules with lightly staining, granular eosinophilic debris, which is derived from exfoliated cortical cells engorged with protein. The basophilic tubules were regarded to be an attempt of the kidney to regenerate. Females of test groups 2 and 3 (30 and 120 mg/kg bw/d) revealed a minimal increase of vacuolation in the kidney that was proven to be fat by ORO-stained slides.
Males of test group 3 (120 mg/kg bw/d) and females of test groups 2 and 3 (30 and 120 mg/kg bw/d) revealed a minimal to slight vacuolation of hepatocytes in the periportal area (zone 1). These vacuoles stained positive with ORO-stain and, therefore, were proven to be fat vacuoles.


OTHER FINDINGS
Estrous cycle:
No test substance-related effects on estrous cycle length and the number of cycles were obtained.

Sperm parameters:
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis treatment-related effects were not observed.

PAL CoA in liver homogenate:
In male and female rats of test group 3 (120 mg/kg bw/d) and additionally in females of test group 2 (30 mg/kg bw/d), cyanide-insensitive Palmitoyl-CoA oxidation in liver tissue was increased. Increases in Palmitoyl-CoA oxidation are a sign for peroxisomal proliferation
Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on peroxisomal proliferation and alpha-2u nephropathy
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
Under the conditions of this study the oral administration i-Nonanoic acid by gavage to Wistar rats over a period of 3 months revealed signs of systemic toxicity at dose levels of 30 and 120 mg/kg bw/d in male and in female animals. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 5 mg/kg bw/d in male and female Wistar rats.
Executive summary:

The test substance i-Nonanoic acid was administered for 3 months daily by gavage to male and female Wistar rats at dose levels of 0, 5, 30 and 120 mg/kg bw/d (test groups 0-3). Corn oil served as vehicle and vehicle control.

Clinical examinations did not reveal treatment-related, adverse effects up to a dose level of 120 mg/kg bw/d. In addition,no test substance-related effects on estrous cycle length and the number of estrous cycles were obtained.

Salivation after treatment was seen in most animals of test group 3 (120 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

Concerning clinical pathology, some affection of the liver cell function could be observed resulting in higher cyanide-insensitive Palmitoyl-CoA oxidation (PAL CoA) in liver tissue of male and female rats in test group 3 (120 mg/kg bw/d) and of females in test group 2 (30 mg/kg bw/d) as well as lower serum albumin levels in females of test group 3 (120 mg/kg bw/d). Higher PAL CoA levels are indicative of an increased β-oxidation of long-chained fatty acids in peroxisomes of liver cells.

Regarding pathology, target organs were the kidney and the liver. The absolute and relative kidney weight of male animals in test group 3 (120 mg/kg bw/d) was significantly increased (15% and 17%, respectively). The relative kidney weight of male animals in test group 2 (30 mg/kg bw/d) was also increased (8%). Males of these test groups revealed eosinophilic droplets (found to be alpha 2u globulin), increased basophilic tubules (regeneration of the tubules) and granular casts (cellular debris within tubules). These findings were responsible for the macroscopically detected light brownish discoloration and the weight increase.Eosinophilic droplets in the proximal convoluted tubules represent alpha 2u globulin, a poorly hydrolysable, low molecular weight protein characteristic for male rats.As secondary effects to the alpha 2u globulin storage in the kidney granular casts and basophilic tubules were observed in males of test group 2 and 3 (30 and 120 mg/kg bw/d; no effects in low dose males). Granular casts are characterized by marked dilation of solitary tubules with lightly staining, granular eosinophilic debris, which is derived from exfoliated cortical cells engorged with protein. The basophilic tubules were regarded to be an attempt of the kidney to regenerate. None of these effects have been observed in female rats; alpha-2u-induced nephropathy is a rat male-specific sign of toxicity.

Therefore, these findings were regarded to be adverse but as this mechanism is only known to occur in the male rat they were regarded not to be of human relevance (Hard et al., 1993).

The fat vacuoles in the liver of males of test group 3 (120 mg/kg bw/d) and the liver and kidneys of females of test groups 2 and 3 (30 and 120mg/kg bw/d) were regarded to be treatment-related. The finding in the clinical pathology (increasePAL CoAoxidation) was regarded to reflect a disturbance in fat metabolism which than consequently lead to an increase in fat storage in liver and kidneys. This was regarded to be adverse. The increase in liver weight was regarded to be in connection with the increase in PAL CoA and, therefore, adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Taken together, peroxisome proliferation and alpha-2u, and secondary effects thereof were the main effects observed in a 90d-repeated dose toxicity study according to OECD TG 408. The NOAEL of the study can be set at the lowest dose, i.e. 5 mg/kg/d, where no treatment-related effects have been observed. However, it should be kept in mind that the findings in the male kidneys were regarded to be adverse but as this mechanism is only known to occur in the rat, they were regarded not to be of human relevance.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
sufficient for evaluation
System:
other: hepatobiliary, urinary
Organ:
kidney
liver

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

Mode of Action Analysis / Human Relevance Framework

see discussion below

Additional information

In a 28 day guideline study the test substance (purity 94.9%) was administered to male and female Wistar rats by gavage for 4 weeks at dose levels of 0 (vehicle control), 10, 50 and 200 mg/kg body weight/day as aqueous solution of 0.1% Cremophor EL. Control and high dose groups consisted of each 10 animals per sex, whereas low and mid dose groups consisted of each 5 animals per sex. 5 animals per sex of all dose groups were sacrificed at the end of exposure (main groups). The remaining 5 animals per sex of control and high dose groups were maintained for another 14 days without administration of the test substance (recovery groups).

Kidney effects were seen in males of all dose groups (dose-related effect). This was attributed to the sex and species-specific hydrocarbon-induced a2u globulin accumulation in the kidneys of male rats and regarded as not relevant for humans. No effects apart from nephrotoxicity in males no effects at all were seen in females at 10 mg/kg bw/d.

At 50 mg/kg bw/d, fatty infiltration of liver cells (4 out of 10 animals) as well as alterations in clinical chemistry parameters and increased kidney weights were observed in females.

At 200 mg/kg bw/d, peripheral fatty infiltration of liver cells was increased in femals (all animals) and observed also in one male. In this dose group the following effects were observed additionally: decreased motor activity (females) and increased liver weights (both sexes) as well as alterations in clinical chemistry and urinalysis parameters (both sexes). Except the kidney lesions in male rats, no treatment-related effects were reported in the recovery group. Despite the effects observed in the 50 mg/kg dose group (fatty degeneration of the liver in female rats) this dose is considered to be the NOAEL:

The minimal liver effects in females at this dose are accompanied by signs of liver peroxisome proliferation, a species-specific effect in rodents. Moreover these effects were completely reversible. Increased kidney weights in females are also reversible and occured without any histological alterations in the kidneys, even at the highest dose tested. Therefore,the NOAEL of this study (BASF, 2002) is considered to be 50 mg/kg bw/d. This well performed guideline study (OECD 407) is considered to be of high reliability (RL1).

Slight maternal effects were observed in a one-generation rat study (reproduction/developmental toxicity screening test), but at far higher doses (Exxon, 1998, see chapter 7.8.1).

The test substance i-Nonanoic acid was administered for 3 months daily by gavage to male and female Wistar rats at dose levels of 0, 5, 30 and 120 mg/kg bw/d (test groups 0-3). Corn oil served as vehicle and vehicle control.

Clinical examinations did not reveal treatment-related, adverse effects up to a dose level of 120 mg/kg bw/d. In addition, no test substance-related effects on estrous cycle length and the number of estrous cycles were obtained.

Salivation after treatment was seen in most animals of test group 3 (120 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

Concerning clinical pathology, some affection of the liver cell function could be observed resulting in higher cyanide-insensitive Palmitoyl-CoA oxidation (PAL CoA) in liver tissue of male and female rats in test group 3 (120 mg/kg bw/d) and of females in test group 2 (30 mg/kg bw/d) as well as lower serum albumin levels in females of test group 3 (120 mg/kg bw/d). Higher PAL CoA levels are indicative of an increased β-oxidation of long-chained fatty acids in peroxisomes of liver cells.

Regarding pathology, target organs were the kidney and the liver. The absolute and relative kidney weight of male animals in test group 3 (120 mg/kg bw/d) was significantly increased (15% and 17%, respectively). The relative kidney weight of male animals in test group 2 (30 mg/kg bw/d) was also increased (8%). Males of these test groups revealed eosinophilic droplets (found to be alpha 2u globulin), increased basophilic tubules (regeneration of the tubules) and granular casts (cellular debris within tubules). These findings were responsible for the macroscopically detected light brownish discoloration and the weight increase. Eosinophilic droplets in the proximal convoluted tubules represent alpha 2u globulin, a poorly hydrolysable, low molecular weight protein characteristic for male rats. As secondary effects to the alpha 2u globulin storage in the kidney granular casts and basophilic tubules were observed in males of test group 2 and 3 (30 and 120 mg/kg bw/d; no effects in low dose males). Granular casts are characterized by marked dilation of solitary tubules with lightly staining, granular eosinophilic debris, which is derived from exfoliated cortical cells engorged with protein. The basophilic tubules were regarded to be an attempt of the kidney to regenerate. None of these effects have been observed in female rats; alpha-2u-induced nephropathy is a rat male-specific sign of toxicity.

Therefore, these findings were regarded to be adverse but as this mechanism is only known to occur in the male rat they were regarded not to be of human relevance (Hard et al., 1993).

The fat vacuoles in the liver of males of test group 3 (120 mg/kg bw/d) and the liver and kidneys of females of test groups 2 and 3 (30 and 120mg/kg bw/d) were regarded to be treatment-related. The finding in the clinical pathology (increasePAL CoAoxidation) was regarded to reflect a disturbance in fat metabolism which than consequently lead to an increase in fat storage in liver and kidneys. This was regarded to be adverse. The increase in liver weight was regarded to be in connection with the increase in PAL CoA and, therefore, adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Taken together, peroxisome proliferation and alpha-2u, and secondary effects thereof were the main effects observed in a 90d-repeated dose toxicity study according to OECD TG 408. The NOAEL of the study can be set at the lowest dose, i.e. 5 mg/kg/d, where no treatment-related effects have been observed. However, it should be kept in mind that the findings in the male kidneys were regarded to be adverse but as this mechanism is only known to occur in the rat, they were regarded not to be of human relevance.


Justification for classification or non-classification

Based on the available results of the key study and according to Regulation (EC) No 1272/2008, no classification is required.