6-[(C10-C13)-alkyl-(branched, unsaturated)-2,5-dioxopyrrolidin-1-yl]hexanoic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-05-08 to 1995-11-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, well-performed and well-documented

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

6-(3-tetrapropenyl-2,5-dioxopyrrolidin-1-yl)hexanoic acid

EC Number:

800-770-5

Cas Number:

1424148-99-1

Molecular formula:

C22H39NO4

IUPAC Name:

6-(3-tetrapropenyl-2,5-dioxopyrrolidin-1-yl)hexanoic acid

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Charles River Company, Ltd.
- Age at study initiation: 15 weeks
- Weight at study initiation: 122.6 to 142.0 g in males and 100.9 to 118.3 g in females
- Fasting period before study:
- Housing: individually in cages with stainless steel wire net flooring in a barrier system rearing room
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 13

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, morning

Doses / concentrations

Remarks:

Doses / Concentrations:
8, 40, 200, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

The study included recovery groups for doses of 200 and 1000 mg/kg bw

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: one or more times a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: one or more times a day

BODY WEIGHT: Yes
- Time schedule for examinations: day -2 before administration , days 1 (administration), 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26 and 28 during the administration period and on days 1(recovery) 3, 5, 8, 10, 12 and 14 during the recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined once before administration and two times a week during the administration and recovery periods.

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 AND COMPOUND INTAKE (if drinking water study): No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: when the administrat.ion period was concluded and when the recovery period was concluded after one night of fasting (16 to 20 hours) .
- Anaesthetic used for blood collection: Yes (ether anesthesia)
- Animals fasted: Yes
- How many animals: all
- Parameters: RBC, WBC, Hb, Ht, MCV, MCH, MCHC, Platelet count, Reticulo, PT, APTT, Differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: when the administrat.ion period was concluded and when the recovery period was concluded after one night of fasting (16 to 20 hours) .
- Animals fasted: Yes
- How many animals: all
- Parameters: GOT, GPT, ALP, ChE, T-Cho, TG, glucose, T-protein, Albumin, A/G ratio, BUN, Creatinine, T-Bil, Ca, IP, Na, K, Cl

URINALYSIS: Yes
- Time schedule for collection of urine: once during the administration period (day, 28) and once during the recovery period (day 14 (recovery).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters: volume, color tone, pH, protein, ketone bodies, bilirubin, occult blood, sugar, urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Test period:
Solvent control and 100 mg/kg group: liver, spleen, kidneys, heart, stomachm intestines, testes, adrenals
200 mg/kg group: Liver, kidneys (males only), stomach (males only), testes
40 mg/kg group :Liver (females only), kidneys (males only)
8 mg/kg group: Kidneys (males only)

Recovery period:
Solvent control group, 1000 mg/kg group: Liver, kidneys (males only), stomach (males only), testes
200 mg/kg group: Liver, kidneys (males only) testes

Other examinations:

Organ weight determinations: brain, liver, spleen, kidney, adrenals, testes (or overies)

Statistics:

The findings for body weight, feed consumption, hematological tests, blood biochemistry tests, urine volume and organ weights were subjected to homoscedastic analysis by Bartlett's test. When equal variance at a 5% level of significance was found, tests between the solvent control group and each administration group were performed by Dunnett'smet.hod when the number of cases in each group were equal and by Scheffe's method when they were not equal.

When equal variance was not found,the Kruskall-Wallis test was performed. When significant differendes were found, tests between the solvent control group and each administration group were performed by Dunnet's method when the number of cases in each group was equal and by Scheffe's nonparametric method when they were not equal.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

no effects observed

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

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 specified

Details on results:

CLINICAL SIGNS AND MORTALITY
at the time administration was concluded:
- no death occurred.
- salivation in males and females in groups given 200 mg/kg and higher
- decrease in spontaneous movement and decrease in respiratory rate in males in the 200 mg/kg group
- decrease in spontaneous movement, decrease in respiratory rate, soiling around the nose and mouth, hunchback posture, soiling around the anus and depilation in the lower neck region in males and females in the 1000 mg/kg group
- soft stool, reddish tears, reddish tear traces and ptosis in males in the 1000 mg/kg group
at the time recovery was concluded:
- No significant effect.

BODY WEIGHT AND WEIGHT GAIN
No effects

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No effects

HAEMATOLOGY
at the time administration was concluded:
-decrease in hemoglobin concentration and in hematocrit values in males and females in the 1000 mg/kg group
- decreases in erythrocyte counts in males in the 1000 mg/kg group
- prolongation of activated partial thromboplastin time and decreases in erythrocyte counts were seen in females in the 1000 mg/kg group
- increases in leukocyte counts in males in groups given 200 mg/kg and higher
- increase in the rod neutrophil ratio in males in the 1000 mg/kg group.
at the time recovery was concluded
- no effects in males
-decreases in erythrocyte counts and homoglobin concentrations and increases in platelet counts in females in 1000 mg/kg bw.

CLINICAL CHEMISTRY
at the time administration was concluded:
- decrease in blood sugar in males in groups given 200 mg/kg and higher
- increase in total bilirubin in males in the 1000 mg/kg group
- decrease in chlorine in females in groups given200 mg/kg and higher
- increase in triglycerides and decrease in creatinine in females in the 1000 mg/kg group
at the ime recovery was concluded
-decrease in creatinine and increase in total bilirubin in males in 200 mg/kg bw and higher.
- no effects in females

ORGAN WEIGHTS
at the time administration was concluded:
-increases in the liver weight in females in groups given 200 mg/kg and higher and in males in the 1000 mg/kg group
at the time recovery was concluded:
-increase in the relative weights of the liver and kidneys in males in the 1000 mg/kg bw group
-increase in the relative weight of the liver in females in the 1000 mg/kg bw group
- increase in the relative weight of the kidneys in females in the 200 mg/kg bw

GROSS PATHOLOGY
at the time administration was concluded:
- apparent surface spotting of the kidneys in males in the 1000 mg/kg group;
- roughening of the mucosa of the glandular stomach in males in the 1000 mg/kg group
- no effect in females
at the time recovery was concluded:
- black spots on the mocosa of the glandular stomach in males in the 200 mg/kg bw group.

HISTOPATHOLOGY
at the time administration was concluded:
- increase in eosinophilic bodies in the kidneys in males in the group given 200 mg/kg and higher
- swelling of hepatocytes in males and females in the 1000 mg/kg group
- granulation tissue accompanied by calcification in the liver in males in the 1000 mg/kg bw
- mucosal degeneration of the proventriculus [forestomach] in males in the 1000 mg/kg group.
at the time recovery was concluded:
- increase in eosinophilic bodies in the liver in males in the 1000 mg/kg bw
-necrosis of the mucosa of the glandular stomach in males in the 200 mg/kg bw

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

40 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table: Reversibility of major findings; histopathology not included
		At the time administration was concluded			At the time recovery was concluded
		Vehicle control	200 mg/kg bw	1000 mg/kg bw	Vehicle control	200 mg/kg bw	1000 mg/kg bw
Hematology
RBC [x10^4/mm^3]	Males	786 ± 14	751 ± 32	716 ** ± 38	829 ± 47	809 ± 19	781 ± 42
	females	738 ± 22	731 ± 36	707 ± 33	794 ± 24	772 ± 21	743 ** ± 23
WBC [x10^2/mm^3]	Males	74 ± 12	108** ± 13	107 ** ±19	117 ± 13	93 ±20	112 ±17
	females	71 ± 18	77 ± 10	82 ± 21	75 ± 30	57 ± 8	73 ± 11
Hb [g/dt]	Males	15.5 ± 0.6	15.0 ± 0.4	14.1 ** ± 0.4	15.6 ± 0.5	15.2 ± 0.7	15.3 ± 0.3
	females	15.1 ± 0.6	14.8 ± 0.6	13.9 ** ± 0.5	15.6 ± 0.4	15.1 ± 0.3	15.0 * 0.5
Ht [%]	Males	44.5 ±1.4	44.0 ± 1.0	41.2 ** ± 1.0	45.0 ± 2.0	43.7 ± 1.6	44.1 ± 1.4
	females	42.1 ± 1.4	41.0 ± 2.5	38.9 * ± 1.5	43.3 ± 0.8	41.9 ± 0.8	42.0 ± 1.5
Platelet [x10^4/mm^3]	Males	121.6 ± 10.9	130.2 ± 15.6	136.4 ± 16.5	115.5 ± 9.9	116.7 ± 10.4	110.6 ± 11.2
	females	129.1 ± 11.6	132.5 16.7	120.2 ± 6.3	120.6 ± 9.4	124.4 ± 11.6	135.7 * ± 6.1
PT [sec]	Males	16.6 ± 3.9	14.9 ± 2.1	17.8 ± 0.9	14.1 ± 1.5	14.5 ± 2.1	15.7 ± 2.5
	females	12.0 ± 0.7	11.5 ± 0.5	11.7 ± 0.9	11.3 ± 0.5	11.0 ± 0.2	10.9 ± 0.3
APTT [sec]	Males	28.9 ± 3.1	30.7 ± 2.9	32.7 3.3	27.2 ± 3.8	24.1 ± 3.4	26.4 ± 1.9
	females	20.7 ± 0.8	22.1 ± 2.1	25.9 ** ± 3.6	21.8 ± 2.3	20.2 ± 3.9	20.2 ± 1.7
Clinical Chemistry
ALP [IU/l]	Males	512 ± 67	490 ± 57	476 ± 67	373 ± 43	336 ± 35	342 ± 40
	females	310 ± 34	265 ± 54	249 ± 31	200 ± 28	196 ± 42	171 ± 27
Glucose  [mg/dl]	Males	133.1 ± 13.4	111.8* 13.8	108.3 * 12.6	150.8 ± 15.6	132.6 ± 16.2	129.7 ± 17.6
	females	116.5 ± 11.1	121.7 ± 14.0	103.8 ± 22.5	124.4 ±12.8	135.9 16.5	127.6 ±10.4
TG [mg/dl]	Males	51 ± 15	66 ±20	58 ±11	63 ± 11	61 ± 13	46 ± 16
	females	29 ± 4	31 ± 7	41 * ± 10	35 ± 11	33 ± 16	31 ± 6
Creatinine [mg/dl]	Males	0.45 ± 0.07	0.39 ± 0.03	0.40 ± 0.05	0.54 ± 0.03	0.47** ± 0.02	0.47 ** ± 0.04
	females	0.46 ± 0.03	0.43 ± 0.04	0.39* ± 0.04	0.50 ± 0.04	0.49 ± 0.05	0.49 ± 0.04
T-Bil [mg/dl]	Males	0.18 ± 0.03	0.22 ± 0.04	0.25 ** 0.04	0.15 ± 0.02	0.18 * ± 0.01	0.19 * ± 0.02
	females	0.18 ± 0.01	0.20 ± 0.02	0.21 ± 0.02	0.17 ± 0.03	0.19 ± 0.04	0.17 ± 0.02
Cl [mEq/l]	Males	106.2 ± 1.5	106.4 ± 1.3	106.0 ± 0.9	106.7 ± 2.0	107.2 ± 1.2	106.8 ± 1.9
	females	109.3 ± 1.0	106.7** ±1.3	106.8** ± 1.1	108.7 ± 1.5	108.4 ± 1.7	108.9 ± 2.6
Relative Organ weight
Liver [g/100g]	Males	3.05 ± 0.22	3.15 ± 0.14	3.81** ± 0.07	2.71 ± 0.05	2.74 ± 0.09	2.98** ± 0.09
	females	3.14 ± 0.18	3.47 * ± 0.17	4.25 ** ± 0.13	2.77 ± 0.12	3.05 ± 0.28	3.08* ± 0.16

Mean ± SD

*: significantly different from vehicle control at p < 0.05

 

Table: Reversibility of histopathological findings (number of animals affected out of six)
			At the time administration was concluded			At the time recovery was concluded
			Vehicle control	200 mg/kg bw	1000 mg/kg bw	Vehicle control	200 mg/kg bw	1000 mg/kg bw
Liver
Swelling of hepatocytes	Males		0	0	3	0	0	0
	females		0	0	4	0	0	0
Kidney
Eosinophilic bodies	males	++	0	3	1	0	0	1
		+++	0	0	3	0	0	0
		++++	0	0	2	0	0	0
	females	++	0	-	0	0	-	-
		+++	0	-	0	0	-	-
		++++	0	-	0	0	-	-
Forestomach
Mucosa degeneration	Males	+	0	0	4	0	-	0
		++	0	0	1	0	-	0
	females	+	0	-	0	-	-	-
		++	0	-	0	-	-	-

+, very slight; ++, slight; +++, moderate; ++++ severe

-; no investigated

Applicant's summary and conclusion

Conclusions:

The registration substance was investigated for its repeated dose toxicity according to the OECD Gudieline 407. Liver was identified as target organ, possibly related to metabolic overload. Further minimal effect on the blood system could be derived. In males, minimal effects o the forestomach and kideny were found additionally. The NOEL of 40 mg/kg bw was obtained.
No classification is warranted with respect to the endpoint repeated dose toxicity.

Executive summary:

The registration substance was investigated according to the OECD Guideline 407. Rats were given via gavage with the test material at doses of 0, 8, 40, 200 and 1000 mg/kg bw. Recovery groups were established for the 1000 and 200 mg/kg groups and the solvent control group.

No deaths occurred. No effects due to administration of the test substance were seen on body weight and feed consumption during the period of administration or on the urinalyses at the time administration was concluded.

Salivation, decrease in spontaneous movement, decrease in respiratory rate, increase in leukocyte counts, decrease in blood sugar, decrease in chlorine, increase in liver weight and increase in eosinophilic bodies in the liver attributable to administration of the test substance were seen in the groups of 200 mg/kg or higher. Further hematology alteration was observed in males and females and effects on forestomach and kidney in males. Most of the effects were either fully reversible within the observation period of 14 days or the degree of severity decreased significantly.

On the basis of the foregoing results, it can be concluded that the principal effects of this substance are on the liver in males and females and on the proventriculus and kidneys in males. It was presumed that the NOEL in rats under the conditions of this test was 40 mg/kg/day.