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Description of key information

For butyric acid, no valid data on repeated dose toxicity could be located.
To compensate for this lack of data, information resulting from n-butyl acetate as supporting substances will be used as substitute. Data are available for inhalation repeated dose toxicity.
Repeated dose toxicity: inhalation
Supporting substance n-butyl acetate: In a valid subchronic (90 d) toxicity study, the NOAEC was determined to be 500 ppm (2400 mg/m³) (OPP/CMA, 1996 and publication David et al., 2001).
The NOAEC for butyric acid (conversion using the respective molecular weights) is ca. 1830 mg/m³.

Key value for chemical safety assessment

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
As working hypothesis for the read-across approch from n-butylacetate to butyric acid, information on toxicokinetics for the metabolization of n-butyl acetate into butanol and acetic acid are used. n-butanol is metabolized in the liver and it undergoes rapid oxidation. n-butyraldehyde and subsequently n-butyric acid is formed.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemicals n-butylacetate was investiagated as a pure compound without significant impurities. The target substance butyric acid is as well regarded and evaluated as pure compound and mono-constituent substance under REACh Legislation. No significant impurities need to be assessed according to analytical data.

3. ANALOGUE APPROACH JUSTIFICATION
For butyric acid, no valid studies concerning repeated dose toxicity could be identified. Available studies show extensive deviations from actual guidelines and were performed with objectives other than to examine substance inherent repeated dose toxicity. Results are not suited to assess the repeated dose toxicity of butyric acid. To compensate for this lack of data, information originating from n-butyl acetate as supporting substance will be used based on following reasons.

After administration, n-butyl acetate will be rapidly metabolized in vivo in blood and various tissues by ubiquitous esterases. Half-life of this transformation is short resulting in complete cleavage of the ester. n-Butanol is formed which is rapidly metabolized in vivo to n-butyraldehyde by alcohol dehydrogenases and subsequently to n-butyric acid by aldehyde dehydrogenases. Thus, in the course of metabolic transformation of n-butyl acetate, butyric acid is generated rapidly and predominant as intermediary metabolite. Thus, it is justified to use n-butyl acetate as supporting substance in the evaluation of the systemic toxic effects of butyric acid.

Effect levels derived for the supporting substance n-butyl acetate will be transformed to effect levels for butyric acid using the respective molecular weights. These values are used to evaluate the toxic potential of butyric acid. The repeated dose toxicity of n-butyl acetate has been investigated in a valid inhalation GLP-study of high reliability (see cross SOURCE reference)

Male and female Sprague-Dawley rats (15 animals/sex/dose group) were exposed to nominal concentrations of 0, 500, 1500 or 3000 ppm of n-butyl acetate for 6 hours per day, 5 days per week for 13 consecutive weeks. The time-weighted average analytical concentrations were within 10% of the target concentrations. Transient signs of sedation were observed during exposure to the 1500 and 3000 ppm concentrations. Body weights were significantly reduced in the mid and high concentration groups. Feed consumption was significantly lower in the 1500 and 3000 ppm group in comparison to the control group. Organ weights affected: weights of liver, kidneys and spleen were significantly lower for the males of the highest concentration goup. Testes and adrenal gland weights for the mid and high concentration groups and the lung weights for the 3000 ppm males were significantly higher than for the control group. Additionally, effects on the stomach (probably stress related) and pulmonary system were observed: Females of the highest concentration group showed signs of irritation of the glandular stomach and necrosis in the non-glandular stomach. Some rats of the 1500 and 3000 ppm group showed degeneration of the olfactory epithelium along the dorsal medial meatus and ethmotubinates of the nasal passages. The severity was mild to moderate for the 3000 ppm group and minimal to mild for the 1500 ppm group.

There was no systemic, organ specific toxicity. The NOAEC for this study is 500 ppm (2400 mg/m³) (OPP/CMA, 1996 and publication David et al., 2001).

Deduction of the NOAEC (inhalation) for Butyric acid

The NOAEC of butyric acid will be calculated on basis of the NOAEC of n-butyl acetate (OPP/CMA, 1996) using the mass concentration/m³ and the respective molecular weights (88.11 and 116.11).

The deduced NOAEC for Butyric acid is 1830 mg/m³ (500 ppm).

Though not valid for assessing the endpoint repeated dose toxicity, some additional information can be derived from available studies. Information presented here may provide an additional understanding of the toxic action of butyric acid.

Objective of the study was to investigate early lesions in the forestomach of rodents (rats, mice, hamsters) caused by short chain fatty acids like propionic acid and butyric acid. These lesions may be related to the production of tumors which were demonstrated to be induced by repeated administration of propionic acid.

After oral administration of butyric acid (4% in diet) to groups of 5 male and female rats, mice and hamsters for 7 days, lesions in the forestomach were observed (acanthosis, edema of the lamina propria and increased numbers of mitotic figures). For rats, acanthosis, epithelial vacuolation and ulceration (with associated marked epithelial hyperplasia) was prevalent. Rats proved to be the most sensitive of the three tested species.

The formation of lesions in the gastro-intestinal tract caused by the oral application of butyric acid is to be expected due to the acidic nature of the test substance. This effect can be understood as local action of the acidic form of the test substance.
Reason / purpose for cross-reference:
read-across source
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Kingston (Stone ridge, NY)
- Age at study initiation: 60 days
- Weight at study initiation: 271 +/- 7 g (males); 215 +/- 8 g (females)
- Fasting period before study: no
- Housing: individually during non expsure periods
- Diet: Certified Rodent Diet (Agway Prolab RMH 3200, ground chow), ad libitum except during exposure
- Water: ad libitum, except during exposure
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature: 67-75°F
- Humidity: 46-60%
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4200 L stainless-steel and glass inhalation chambers
- Method of holding animals in test chamber: cages
- System of generating vapour: test substance was metered into glass distillation columns packed with glass beads; filtered, compressed air was passed through the glass bead-packed columns to evaporate the test substance; distillation columns were heated to about 50°C to enhance vaporization; the resultant vapour was directed via glass tubing to a tee just upstream of the inhalation chamber where it was mixed with filtered, conditioned outside air
- Temperature, humidity in air chamber: 21.1-24.7°C; 36.7-68.7%
- Air flow rate: 836 to 965 Lpm
- Air change rate: 12 to 14 air changes per hour
- Method of particle size determination: Micro Laser Particle counter (µLPC-301, Particle Measuring Systems, Inc, Coulder, USA); indicating that an aerosol fo the test subsance was not present



TEST ATMOSPHERE
- Brief description of analytical method used: MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) set at a wavelength of 3.38 µM
- Samples taken from breathing zone: no; collection of chamber vapour samples

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) set at a wavelength of 3.38 µM
- chamber vapour samples were continuously collected from each chamber throught TEFLON tubing (3/16" i.d.)
- valve position was pepriodically changed to sample from each chamber at least once each hour
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Dose / conc.:
500 ppm (nominal)
Dose / conc.:
1 500 ppm (nominal)
Dose / conc.:
3 000 ppm (nominal)
No. of animals per sex per dose:
15
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Range finding study: 2-Weeks repeated exposure in which animals were exposed to 0, 750, 1500 or 3000 ppm n-butyl acetate. The test substance produced concentration-related reductions in general activity levels during exposure periods. Animals appeared to acclimate to the 750 and 1500 ppm concentrations but not to 3000 ppm. Mean body weights for the female 1500 ppm animals and for the 3000 ppm male and female animals were lower than the control group on Days 7 and 14, but no statistically significant differences were noted. 3000 ppm was selected as an exposure concentration that would produce overt signs of toxicity, and 500 ppm was selected as an exposure concentration that was expected to have no effect. An exposure concentration of 1500 ppm was selected as the intermediate exposure concentration.
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during exposure and once per day on weekends
- Cage side observations checked were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before and after exposure


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION: YES
- Time schedule for examinations: weekly

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: No


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of the study and during the last week of exposure
- Dose groups that were examined: all animals prior to the start of the study; animals from the control and high-concentration group during the last week of exposure


HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 30 and 90 of the study
- Anaesthetic used for blood collection: Yes (Metofane)
- Animals fasted: Yes
- How many animals: 5 per sex per dose group
- Parameters checked:
Whole blood: hemoglobin concentration, red blood cell count, white blood cell count, hematocrit, red blood cell indices, prothrombin time
Blood smears: cellular morphology and differential white blood cell count


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 30 and 90 of the study
- Anaesthetic used for blood collection: Yes (Metofane)
- Animals fasted: Yes
- How many animals: 5 per sex per dose group
- Parameters checked:
Blood serum: aspartate aminotransferase, sorbitol dehydrogenase, alkaline phosphatase, creatinine, albumin, total protein, total bilirubin, alanine aminotransferase, gamma glutamyltranspeptidase, urea nitrogen, glucose, calcium, phosphorus


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Organ weights (liver, kidneys, testes or ovaries, spleen adrenal glands, lungs, brain)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (embedded in paraffin, sectioned at 5 µm and stained with hematoxylin and eosin; nasal passages were decalcified prior to being embedded and sectioned); all tissues (see below) were examined microscopically from the control and high-concentration groups, in addition, the lungs, nasal passages, thymus (males only), stomach (females only), and gross lesions were examined from the mid- and low concentration group
- tissues examined: nasal passages, trachea, larynx, lungs, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, aorta, adrenal glands, pituitary gland, thymus, pancreas, urinary bladder, thyroid gland, parathyroid gland, spleen, liver, kidneys, mesenteric lymph nodes, sternum (with bone marrow), right testis, right epididymis, male accessory sex glands, ovaries, vagina, uterus, fallopian tubes, salivary glands, gross lesions
Other examinations:
- assessment of sperm morphology and development (reported in section 7.8)
Statistics:
Bartlett's test, one-way analysis of variance, duncan's multiple range test, Kruskal-Wallis H-test and Mann-Whitney U-test
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- no spontaneous mortality occurred during the study

3000 ppm group:
- reduced activity levels of generally minor severity during exposure (less movement, decreased alertness, slower response to tapping on the chamber wall)
- Sialorrhea was observed in three animals for no more than one or two exposure days
- red discoloration of the chin hair occurred in several females for no more than one or two exposure days

1500 ppm group:
- reduced activity of generally minimal severity during exposure periods (exceüpt the first 5 hours of Day 0 and the first hour or two of Days 1 and 2)

Animals in all groups had prophyrin nasal discharges and dried porphyrin stains around the nose, which occasionally persisted till the next morning. Animals from all groups exhibited discolored-red facial hair, usually the chin hair (slightly higher in the 3000 ppm group than in other groups)

BODY WEIGHT AND WEIGHT GAIN

3000 ppm group:
- significantly reduced mean body weights and body weight gains in males and females; overall weight gains were 62% and 78% of weight gains for the control group (males and females, respectively)

1500 ppm group:
- significantly reduced mean body weights and body weight gains in males and females; overall weight gains were 77% and 70% of weight gains for the control group (males and females, respectively)

500 ppm group:
- body weights and body weight gains were not affected; overall weight gains were 90% and 107% of weight gains for the control group (males and females, respectively)

FOOD CONSUMPTION

3000 ppm group:
- significantly lower than for the control group
- mean weekly feed consumption values were 14-25% or 6-16% lower than the control group in male and female rats, respectively

1500 ppm group:
- significantly lower than for the control group
- mean weekly feed consumption values were 4-17% or 10-15% lower than the control group in male and female rats, respectively
500 ppm group:
- significantly (p - mean weekly feed consumption values were 3-12% lower than the control group in male rats and from 2% higher to 7% lower than the control group for female rats
FOOD EFFICIENCY
- no data

WATER CONSUMPTION
- no data

OPHTHALMOSCOPIC EXAMINATION
- no effects

HAEMATOLOGY
No significant differences in hematologic parameters were seen after 30 days on test. Significantly higher (p Evaluation of blood cell morphology did not suggest any compound-related effects. After 30 days on test, spherocytosis and poikilocytosis were seen in blood smears of animals from most groups. Increased polychromasia was observed in one male control rat (# 613), while decreased polychromasia was see in two female 3000 ppm rats (# 719 and 720). Howell-Jolly bodies in the blood and anisocytosis were also noted for Rat # 720. After 90 days on test, poikilocytosis was seen in animals from all groups. Anisocytosis was noted in animals from the control, 500, and 1500 ppm groups. Microcytosis was seen in one male 1500 ppm rat (# 632) and in one male 500 ppm rat (#624) and spherocytosis was seen in two male 1500 ppm rats (# 631 and 638).

CLINICAL CHEMISTRY
After 30 days on test, mean sodium concentrations for the male and female 3000 ppm groups were significantly lower (p After 90 days on test, mean albumin and total protein concentrations for the 3000 ppm female group were significantly lower (p
URINALYSIS
- not examined

NEUROBEHAVIOUR
- not examined

ORGAN WEIGHTS
Mean terminal body weights measured after exsanguination were significantly lower (p kidney weights for the 1500 female and 3000 ppm male and female groups were also significantly lower (p

GROSS PATHOLOGY
- no exposure related effects were observe in male rats.
- Two females of the 3000 ppm group showed hemorrhage involving the glandular stomach (minimal severity). White discoloration in the non-glandular stomach was also observe for these animals.
- No changes were seen in female rats from the 1500 and 500 ppm groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
- exposure related changes were observed in the nasal passages and stomach of 1500 and 3000 ppm rats
- all male and female 3000 ppm rats and 4/10 male and 6/10 female 1500 ppm rats had necrosis of the olfactory epithelium (mild to moderate for the 3000 ppm group, mild for the 1500 ppm group).
- no lesions were observed in the nasal passages of the 500 ppm group
- 3/10 female rats of the 3000 ppm group had inflammation of the stomach mucosa (glandular or forestomach) of mild to minimal severity; the pathologist concluded that this effect was probably due to stress
- no effects were seen in the low- and mid concentration group
- thymus atrophy was observed in one male of the 3000 ppm group, this lesion was attributed to stress by the pathologist

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
- not examined


OTHER FINDINGS
- No exposure-related effect on epidydimidal or testicular sperm count was observed. Effects on testicular staging or spermatogenic were not evaluated due to the unacceptable condition of tissue.
Key result
Dose descriptor:
NOAEC
Effect level:
1 830 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

- overall time-weighted average analytical concentrations:

548.4; 1487.5, 3009.6 ppm for males

547.9, 1487.6, 3008.8 ppm for females

- target analytical concentration for the 500 ppm group was increased to 550 ppm to ensure that exposure concentration was at least 500 ppm at all locations of the eposure chamber

Conclusions:
Subchronic exposure of rats to n-butyl acetate vapour resulted in acute, transient signs of reduced activity levels during exposure to 1500 and 3000 ppm. Decreased body weight and feed consumption were noted for the 1500 and 3000 ppm groups, but there was no systemic or organ-specific toxicity. Signs of upper respiratory tract irritation were seen in the nasal passages of 1500 and 3000 ppm animals, but there was no evidence of pulmonary toxicity. The no-observed-adverse-effect concentration (NOAEC) for this study is considered to be 500 ppm (2.4 mg/L).
Executive summary:

Male and female Sprague-Dawley rats (15 animals/sex/dose group) were exposed to nominal concentrations of 0, 500, 1500 or 3000 ppm of n-butyl acetate for 6 hours per day, 5 days per week for 13 consecutive weeks. The time-weighted average analytical concentrations were within 10% of the target concentrations. Transient signs of sedation were observed during exposure to the 1500 and 3000 ppm concentrations. Body weights were significantly reduced in the mid and high concentration groups. Feed consumption was significantly lower in the 1500 and 3000 ppm group in comparison to the control group. Organ weights affected: weights of liver, kidneys and spleen were significantly lower for the males of the highest concentration goup. Testes and adrenal gland weights for the mid and high concentration groups and the lung weights for the 3000 ppm males were significantly higher than for the control group. Additionally, effects on the stomach (probably stress related) and pulmonary system were observed: Females of the highest concentration group showed signs of irritation of the glandular stomach and necrosis in the non-glandular stomach. Some rats of the 1500 and 3000 ppm group showed degeneration of the olfactory epithelium along the dorsal medial meatus and ethomtubinates of the nasal passages. The severity was mild to moderate for the 3000 ppm group and minimal to mild for the 1500 ppm group. There was no systemic, organ specific toxicity. The no-observed-adverse-effect concentration (NOAEC) for this study is 500 ppm (2.4 mg/L) (Bernard and David, 1996; David et al., 2001).

The NOAEC for butyric acid (conversion using the respective molecular weights) is ca. 1830 mg/m³.

Endpoint conclusion
Dose descriptor:
NOAEC
1 830 mg/m³

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For butyric acid, no valid studies concerning repeated dose toxicity could be identified. Available studies show extensive deviations from actual guidelines and were performed with objectives other than to examine substance inherent repeated dose toxicity. Results are not suited to assess the repeated dose toxicity of butyric acid.

 

To compensate for this lack of data, information originating from n-butyl acetate as supporting substance will be used based on following reasons.

 

After administration, n-butyl acetate will be rapidly metabolized in vivo in blood and various tissues by ubiquitous esterases. Half-life of this transformation is short resulting in complete cleavage of the ester. n-Butanol is formed which is rapidly metabolized in vivo to n-butyraldehyde by alcohol dehydrogenases and subsequently to n-butyric acid by aldehyde dehydrogenases. Thus, in the course of metabolic transformation of n-butyl acetate, butyric acid is generated rapidly and predominant as intermediary metabolite. Thus, it is justified to use n-butyl acetate as supporting substance in the evaluation of the systemic toxic effects of butyric acid.

 

Effect levels derived for the supporting substance n-butyl acetate will be transformed to effect levels for butyric acid using the respective molecular weights. These values are used to evaluate the toxic potential of butyric acid.

 

The repeated dose toxicity of n-butyl acetate has been investigated in a valid inhalation GLP-study of high reliability (RL1). This will be the key study

 

Supporting substance: n-butyl acetate

 

Male and female Sprague-Dawley rats (15 animals/sex/dose group) were exposed to nominal concentrations of 0, 500, 1500 or 3000 ppm of n-butyl acetate for 6 hours per day, 5 days per week for 13 consecutive weeks. The time-weighted average analytical concentrations were within 10% of the target concentrations. Transient signs of sedation were observed during exposure to the 1500 and 3000 ppm concentrations. Body weights were significantly reduced in the mid and high concentration groups. Feed consumption was significantly lower in the 1500 and 3000 ppm group in comparison to the control group. Organ weights affected: weights of liver, kidneys and spleen were significantly lower for the males of the highest concentration goup. Testes and adrenal gland weights for the mid and high concentration groups and the lung weights for the 3000 ppm males were significantly higher than for the control group. Additionally, effects on the stomach (probably stress related) and pulmonary system were observed: Females of the highest concentration group showed signs of irritation of the glandular stomach and necrosis in the non-glandular stomach. Some rats of the 1500 and 3000 ppm group showed degeneration of the olfactory epithelium along the dorsal medial meatus and ethmotubinates of the nasal passages. The severity was mild to moderate for the 3000 ppm group and minimal to mild for the 1500 ppm group.

 

There was no systemic, organ specific toxicity.

 

The NOAEC for this study is 500 ppm (2400 mg/m³) (OPP/CMA, 1996 and publication David et al., 2001).

 

Deduction of the NOAEC (inhalation) for Butyric acid

 

The NOAEC of butyric acid will be calculated on basis of the NOAEC of n-butyl acetate (OPP/CMA, 1996) using the mass concentration/m³ and the respective molecular weights (88.11 and 116.11).

 

The deduced NOAEC for Butyric acid is 1830 mg/m³ (500 ppm).

 

Though not valid for assessing the endpoint repeated dose toxicity, some additional information can be derived from available studies. Information presented here may provide an additional understanding of the toxic action of butyric acid.

 

Harrison 1991

 

Objective of the study was to investigate early lesions in the forestomach of rodents (rats, mice, hamsters) caused by short chain fatty acids like propionic acid and butyric acid. These lesions may be related to the production of tumors which were demonstrated to be induced by repeated administration of propionic acid.

 

After oral administration of butyric acid (4% in diet) to groups of 5 male and female rats, mice and hamsters for 7 days, lesions in the forestomach were observed (acanthosis, edema of the lamina propria and increased numbers of mitotic figures). For rats, acanthosis, epithelial vacuolation and ulceration (with associated marked epithelial hyperplasia) was prevalent. Rats proved to be the most sensitive of the three tested species.

 

The formation of lesions in the gastro-intestinal tract caused by the oral application of butyric acid is to be expected due to the acidic nature of the test substance. This effect can be understood as local action of the acidic form of the test substance.

Justification for classification or non-classification

It is concluded that no classification for repeated dose toxicity is necessary because adverse effects after repeated exposure were only observed at doses above the guidance values for classification.

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