Butanal, reaction products with aniline

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Butanal, Reaction Products with Aniline was administered by gavage to 5 male and 5 female Wistar (HsdRCCHan) rats per dose group in daily doses of 0, 25, 100 or 400 mg/kg body weight for a period of at least 32 days. As vehicle corn oil was used. The administration volume was 5 mL/kg.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Butanal, reaction products with aniline

EC Number:

270-109-8

EC Name:

Butanal, reaction products with aniline

Cas Number:

68411-20-1

IUPAC Name:

aniline; butanal

Test material form:

other: liquid

Details on test material:

Test Substance/Item
Test Item Butanal, reaction products with aniline
Aniline content 0,4%
CAS No. 68411-20-1
EC-No. 270-109-8
Chemical name Butanal, reaction products with aniline
CAS name Butanal, reaction products with aniline
Purity 96,6% (taken as 100%)
Appearance Liquid, yellow
Storage Room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

at least 32 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 25, 100 or 400 mg/kg body weight
Basis:
other: nominal

No. of animals per sex per dose:

5 male and 5 female rats/dose

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

The animals were regularly observed and weighed and food and water intakes were determined. In addition, clinical pathology of blood samples was performed.

Sacrifice and pathology:

Organs and tissues were subjected to gross and histopathological investigation.

Other examinations:

Functional observational battery (FOB) and motor and locomotor activity measurements (MA/LMA) were done.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Detailed clinical observations and in-cage observations revealed increased salivation in females starting at 1 00 mg/kg and in males at 400 mg/kg. In addition, distended abdomen was found in both sexes at 400 mg/kg. In addition, signs of impaired general condition (curved back, labored breathing, bloody nose, bloody muzzle) were seen in some male animals at 400 mg/kg. Furthermore, females showed dark-yellowish urine.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights were reduced significantly in male rats at 400 mg/kg and a trend was seen at 100 mg/kg. Females did not show any effects.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake was comparable to controls at the doses of25 and 100 mg/kg in males and in all dosed females. At the high dose of 400 mg/kg in males, a decrease in food intake (-15.7%) was found.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

The water intake of males was not altered up to the dose of 100 mg/kg; at 400 mg/kg the water intake was increased. In females, water intake was increased at 1 00 and 400 mg/kg.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology revealed statistically significant changes in red blood parameters in both sexes at 100 mg/kg and/or 400 mg/kg: ERY, HB, HCT, MCHC were decreased in both sexes. MCH,MCV, and RETI increased in both sexes. In addition, THRO was increased in both sexes and Hep-Quick decreased in females only. For some of the parameters, a trend was already found at 25 mg/kg, however, the difference to controls was in the range of physiological adaptive reactions. In white cell parameters no relevant effect occurred.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical chemistry showed an increase of UREA and an increase of K at 400 mg/kg in both sexes. In addition, an increase in ALA T was found in both sexes at 400 mg/kg which is considered as adaptive change due to liver weight increase. APh (starting at 25 mg/kg), GLUCOSE (starting at 100 mg/kg), CHOL (starting at 100 mg/kg) were lower in both sexes and CREA was lower in females ( 400 mg/kg). These findings are indicators of changes in metabolism, possibly related to the reduced food intake and the lower body weight gain.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

An increase in ALA T was found in both sexes at 400 mg/kg which is considered as adaptive change due to liver weight increase. APh (starting at 25 mg/kg), GLUCOSE (starting at 100 mg/kg), CHOL (starting at 100 mg/kg) were lower in both sexes and CREA was lower in females ( 400 mg/kg). These findings are indicators of changes in metabolism, possibly related to the reduced food intake and the lower body weight gain.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observations revealed no toxicological relevant effects up to 400 mg/kg. Body temperature was slightly decreased at 100 and 400 mg/kg in females and rearing was decreased at 400 mg/kg in females. Male animals were not affected. The activity determination over the entire 60-minute observation period revealed no statistically significant effect on motor (MA) and locomotor activity (LMA) up to 400 mg/kg.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights were changed as follows: Adrenals, heart, liver and spleen weights were increased in both sexes at 400 mg/kg. Thymus weights were decreased in both sexes at 400 mg/kg.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy revealed discoloration of the spleen in all animals at 100 and 400 mglkg and organ enlargement at 400 mg/kg. Discoloration of the liver and kidneys occurred in single animals of both genders at 400 mg/kg. These findings corresponded to an increased blood amount in the spleen and/or to pigment deposition in the named organs.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology showed changes in various organs:
- Spleen: Increased extramedullary hematopoiesis starting at 25 mg/kg in males and at 400 mg/kg in females; increased pigment deposition starting at 25 mg/kg in females and at 100 mg/kg in males; sinus congestion starting at 25 mg/kg; thickening of the splenic capsule starting at 100 mg/kg; Prussian blue stain showed increased iron deposition at 25 mg/kg and above. The extent of the changes at 25 mg/kg was minimal and is
considered in the range of physiological adaptive reactions.
- Liver: Hepatocellular hypertrophy at 100 mg/kg (males only) and at 400 mg/kg in both sexes; increased hepatocellular vacuolation at 400 mg/kg; Oil Red 0 stain showed an increased accumulation of fat in hepatocytes at 400 mg/kg and Prussian blue stain revealed increased iron deposition in Kupffer cells at 400 mg/kg.
- Heart: Myocardial vacuolation starting at 100 mg/kg; myocardial hypertrophy and mononuclear cell infiltration at 400 mg/kg.
- Bone marrow (femur and sternum): Increase in cellularity starting at 100 mg/kg.
- Thyroid gland: Hyertrophy/hyperplasia of follicular cell epithelia at 100 mg/kg and above.
- Kidneys: Tubular swelling/degeneration at 100 mg/kg and above (males only); increased pigment deposition at 100 mg/kg (females only) and at 400 mg/kg in both sexes.
- Adrenals: Increased vacuolation of zona fasciculata cells at 100 mg/kg (males only) and 400 mg/kg in both genders.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Under the conditions described above, a NOEL (no-observed- effect-level) for Butanal, Reaction Products with Aniline after 4-week daily oral treatment by gavage cannot be established for both sexes due to the findings at the low dose of 25 mg/kg affecting red blood cells and spleen. As the extent of the findings is in the range of physiological adaptive reactions, the low dose of 25 mg/kg is still regarded as NOAEL. At 100 mg/kg and higher, clear-cut adverse effects were identified affecting also other organs. In addition, signs of general toxicity occurred.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

other: Spleen, liver, heart, bone marrow, thyroid gland, kidneys, adrenals

Organ:

adrenal glands

bone marrow

heart

kidney

liver

spleen

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

All animals survived until scheduled necropsy.

Detailed clinical observations and in-cage observations revealed increased salivation in females starting at 100 mg/kg and in males at 400 mg/kg. In addition, distended abdomen was found in both sexes at 400 mg/kg. In addition, signs of impaired general condition (curved back, labored breathing, bloody nose, bloody muzzle) were seen in some male animals at 400 mg/kg. Furthermore, females showed dark-yellowish urine.

Functional observations revealed no toxicological relevant effects up to 400 mg/kg. Body temperature was slightly decreased at 100 and 400 mg/kg in females and rearing was decreased at 400 mg/kg in females. Male animals were not affected. The activity determination over the entire 60-minute observation period revealed no statistically significant effect on motor (MA) and locomotor activity (LMA) up to 400 mg/kg.

Body weights were reduced significantly in male rats at 400 mg/kg and a trend was seen at 100 mg/kg. Females did not show any effects.

Food intake was comparable to controls at the doses of 25 and 100 mg/kg in males and in all dosed females. At the high dose of 400 mg/kg in males, a decrease in food intake (-15.7%) was found.

The water intake of males was not altered up to the dose of 100 mg/kg; at 400 mg/kg the water intake was increased. In females, water intake was increased at 100 and 400 mg/kg.

Hematology revealed statistically significant changes in red blood parameters in both sexes at 100 mg/kg and/or 400 mg/kg: ERY (erytrocytes), HB (hemoglobin), HCT (hematocrit), MCHC (mean corpuscular hemoglobin concentration) were decreased in both sexes. MCH (mean corpuscular hemoglobine), MCV (mean corpuscular volume), and RETI (reticulocytes) increased in both sexes. In addition, THRO (platelets/thrombocytes) was increased in both sexes and Hep-Quick decreased in females only. For some of the parameters, a trend was already found at 25 mg/kg, however, the difference to controls was in the range of physiological adaptive reactions.

In white cell parameters no relevant effect occurred.

Clinical chemistry showed an increase of UREA and an increase of K at 400 mg/kg in both sexes.

In addition, an increase in ALAT was found in both sexes at 400 mg/kg which is considered as adaptive change due to liver weight increase. APh (starting at 25 mg/kg), GLUCOSE (starting at 100 mg/kg), CHOL (starting at 100 mg/kg) were lower in both sexes and CREA was lower in females (400 mg/kg). These findings are indicators of changes in metabolism, possibly related to the reduced food intake and the lower body weight gain.

Organ weights were changed as follows: Adrenals, heart, liver and spleen weights were increased in both sexes at 400 mg/kg. Thymus weights were decreased in both sexes at 400 mg/kg.

Necropsy revealed discoloration of the spleen in all animals at 100 and 400 mg/kg and organ enlargement at 400 mg/kg. Discoloration of the liver and kidneys occurred in single animals of both genders at 400 mg/kg. These findings corresponded to an increased blood amount in the spleen and/or to pigment deposition in the named organs.

Histopathology showed changes in various organs:

-       Spleen: Increased extramedullary hematopoiesis starting at 25 mg/kg in males and at 400 mg/kg in females; increased pigment        deposition starting at 25 mg/kg in females and at 100 mg/kg in males; sinus congestion starting at 25 mg/kg; thickening of the        splenic capsule starting at 100 mg/kg; Prussian blue stain showed increased iron deposition at 25 mg/kg and above. The extent        of the changes at 25 mg/kg was minimal and is considered in the range of physiological adaptive reactions.

-       Liver: Hepatocellular hypertrophy at 100 mg/kg (males only) and at 400 mg/kg in both sexes; increased hepatocellular               vacuolation at 400 mg/kg; Oil Red O stain showed an increased accumulation of fat in hepatocytes at 400 mg/kg and Prussian        blue stain revealed increased iron deposition in Kupffer cells at 400 mg/kg.

-       Heart: Myocardial vacuolation starting at 100 mg/kg; myocardial hypertrophy and mononuclear cell infiltration at 400 mg/kg

-       Bone marrow (femur and sternum): Increase in cellularity starting at 100 mg/kg

-       Thyroid gland: Hyertrophy/hyperplasia of follicular cell epithelia at100 mg/kg and above

-       Kidneys: Tubular swelling/degeneration at 100 mg/kg and above (males only); increased pigment deposition at 100 mg/kg               (females only) and at 400 mg/kg in both sexes

-       Adrenals: Increased vacuolation of zona fasciculata cells at 100 mg/kg (males only) and 400 mg/kg in both genders

Applicant's summary and conclusion

Conclusions:

Under the conditions described above, a NOEL (no-observed- effect-level) for Butanal, Reaction Products with Aniline after 4-week daily oral treatment by gavage cannot be established for both sexes due to the findings at the low dose of 25 mg/kg affecting red blood cells and spleen. As the extent of the findings is in the range of physiological adapative reactions, the low dose of 25 mg/kg is still regarded as NOAEL. At 100 mg/kg and higher,
clear-cut adverse effects were identified affecting also other organs. In addition, signs of general toxicity occurred.

Executive summary:

Butanal, Reaction Products with Aniline was administered by gavage to 5 male and 5 female Wistar (HsdRCCHan) rats per dose group in daily doses of 0, 25, 100 or 400 mg/kg body weight for a period of at least 32 days. As vehicle cornoil was used. The administration volume was 5 mL/kg.

The animals were regularly observed and weighed and food and water intakes were determined. In addition, clinical pathology of blood samples was performed. Organs and tissues were subjected to gross and histopathological investigation. Functional observational battery (FOB) and motor and locomotor activity measurements (MA/LMA) were done.

Test substance formulations were stable during the period of use. Content checks during the study revealed correct concentrations of the test substance in the formulations used.

All animals survived until scheduled necropsy.

Detailed clinical observations and in-cage observations revealed increased salivation in females starting at 100 mg/kg and in males at 400 mg/kg. In addition, signs of impaired general condition were seen in some male animals at 400 mg/kg. Furthermore, females showed dark-yellowish urine.

Functional observations revealed no toxicological relevant effects up to 400 mg/kg.

Body weights were reduced significantly in male rats at 400 mg/kg and a trend was seen at 100 mg/kg. Females did not show any effects.

Food intake was comparable to controls at the doses of 25 and 100 mg/kg in males and in all dosed females.

The water intake of males was not altered up to the dose of 100 mg/kg.

Hematology revealed statistically significant changes in red blood parameters in both sexes at 100 mg/kg and/or 400 mg/kg. In white cell parameters no relevant effect occurred.

Clinical chemistry showed an increase of UREA and an increase of K at 400 mg/kg in both sexes.

In addition, an increase in ALAT was found in both sexes at 400 mg/kg which is considered as adaptive change due to liver weight increase. APh (starting at 25 mg/kg), GLUCOSE (starting at 100 mg/kg), CHOL (starting at 100 mg/kg) were lower in both sexes and CREA was lower in females (400 mg/kg). These findings are indicators of changes in metabolism, possibly related to the reduced food intake and the lower body weight gain.

Organ weights were changed as follows: Adrenals, heart, liver and spleen weights were increased in both sexes at 400 mg/kg. Thymus weights were decreased in both sexes at 400 mg/kg.

Necropsy revealed discoloration of the spleen in all animals at 100 and 400 mg/kg and organ enlargement at 400 mg/kg. Discoloration of the liver and kidneys occurred in single animals of both genders at 400 mg/kg. These findings corresponded to an increased blood amount in the spleen and/or to pigment deposition in the named organs.

Histopathology showed changes in various organs:

-       Spleen: Increased extramedullary hematopoiesis starting at 25 mg/kg in males and at 400 mg/kg in females; increased pigment deposition starting at 25 mg/kg in females and at 100 mg/kg in males; sinus congestion starting at 25 mg/kg; thickening of the splenic capsule starting at 100 mg/kg; Prussian blue stain showed increased iron deposition at 25 mg/kg and above. The extent of the changes at 25 mg/kg was minimal and is considered in the range of physiological adaptive reactions.

-       Liver: Hepatocellular hypertrophy at 100 mg/kg (males only) and at 400 mg/kg in both sexes; increased hepatocellular vacuolation at 400 mg/kg; Oil Red O stain showed an increased accumulation of fat in hepatocytes at 400 mg/kg and Prussian blue stain revealed increased iron deposition in Kupffer cells at 400 mg/kg.

-       Heart: Myocardial vacuolation starting at 100 mg/kg; myocardial hypertrophy and mononuclear cell infiltration at 400 mg/kg

-       Bone marrow (femur and sternum): Increase in cellularity starting at 100 mg/kg

-       Thyroid gland: Hyertrophy/hyperplasia of follicular cell epithelia at100 mg/kg and above

-       Kidneys: Tubular swelling/degeneration at 100 mg/kg and above (males only); increased pigment deposition at 100 mg/kg (females only) and at 400 mg/kg in both sexes

-       Adrenals: Increased vacuolation of zona fasciculata cells at 100 mg/kg (males only) and 400 mg/kg in both genders

Conclusion:

Under the conditions described above, a NOEL (no-observed- effect-level) for Butanal, Reaction Products with Aniline after 4-week daily oral treatment by gavage cannot be established for both sexes due to the findings at the low dose of 25 mg/kg affecting red blood cells and spleen. As the extent of the findings is in the range of physiological adapative reactions, the low dose of 25 mg/kg is still regarded as NOAEL. At 100 mg/kg and higher, clear-cut adverse effects were identified affecting also other organs. In addition, signs of general toxicity occurred.