Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study according to guideline.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(March 22, 1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (July 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid, colorless, clear
Details on test material:
- Name of test material: Delta-Valerolactone

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: about 11-12 weeks old
- Weight at study initiation:
- Housing: 1 animal per cage. Exceptions: during mating 1 male/1 female per cage, during rearing up to PND 4:1 dam with her litter
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Air changes: 15 per hour
- Photoperiod: 12 h light / 12 h darkness (6:00 to 18:00 h)

IN-LIFE DATES: From: 04-Sep-2012 To: 05-Nov-2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Delta-Valerolactone 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 prepared daily. The administration volume was 4 mL/kg body weight.

VEHICLE
- Justification for use and choice of vehicle: standard vehicle for studies of this type
- Concentration in vehicle: 25, 75, 250 mg/mL
- Amount of vehicle: 4 mL/kg bw (dose volume)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany, as part of this study.

The stability of Delta-Valerolactone in corn oil at room temperature for a period of 7 days was demonstrated before the start of the study. To ensure the content of the test substance in the vehicle, the test substance preparations were prepared daily.

At the beginning of the study each 6 samples were taken from the lowest and highest concentration for homogeneity analyses. These samples were used as a concentration control at the same time. Sampling was done under administration conditions out of the Erlenmeyer flasks (each 2 samples from bottom, mid, and top of the beaker). Two samples from the mid concentration were taken for concentration control analysis. Each one sample was analyzed in the analytical laboratory, the other was kept frozen at the laboratory Subchronic/Chronic Tox. Rodents until finalization of the report.

Duration of treatment / exposure:
Males:
- 14 days premating
- up to 14 days mating
- sacrifice minimum 4 days after littering
The exposure duration was at least 36 days.

Females:
- 14 days premating
- up to 14 days mating
- gestation about 22 days
-sacrifice minimum 4 days after littering
The exposure duration was at least 56 days.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 or 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 males/ 10 females
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION: Yes
- Time schedule: Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14, 14-20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

WATER CONSUMPTION: Yes
- Time schedule for examinations: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.
- Anaesthetic used for blood collection: Yes, the animals were anaesthetized using isoflurane (Isoba, Essex GmbH, Munich, Germany).
- Animals fasted: Yes
- How many animals: Examinations were carried out in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.
- Parameters examined. Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET), Prothrombin time (HQT). In addition clotting tests were performed.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.
- Animals fasted: Yes
- How many animals: Examinations were carried out in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA).

URINALYSIS: Yes
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, Turbidity, Volume

NEUROBEHAVIOURAL EXAMINATIONS
FUNCTIONAL OBSERVATION BATTERY: Yes
- Time schedule: A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the rats were transferred to single-animal polycarbonate cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

MOTOR ACTIVITY ASSESSMENT: Yes
- Time schedule: Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the rats were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the rats were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the rats during these measurements and the measurement room was darkened after the transfer of the last rat.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS
- Organ weights: epididymides, testes (all male animals); adrenals, brain, heart, kidneys, liver, spleen, thymus (5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations)).

PRESERVATION OF TISSUES
Tissues preserved: adrenals, aorta, bone marrow (femur), brain, cecum, cervix, coagulating gland, colon, duodenum, epididymides, esophagus, eyes with optic nerve, extraorbital lacrimal glands, female and male mammary gland, femoral bone with articulation, heart, ileum, jejunum (with Peyers patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid gland, pharynx, pituitary gland, prostate, rectum, salivary glands (sublingual and mandibular), sciatic nerves, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach and glandular stomach, target organs, testes, thymus, thyroids, trachea, urinary bladder, uterus, vagina, all gross lesions

HISTOPATHOLOGY: Yes, Mainly 5 animals/dose group from the control and high dose group, except all gross lessions and liver (all dose groups).
- Organs examined: adrenals, bone marrow (femur), brain, cecum, cervix, coagulating gland, colon, duodenum, epididymides, heart, ileum, jejunum (with Peyers patches), kidneys, liver, lungs, lymph nodes (axillary and mesenteric), ovaries, oviducts, prostate, rectum, sciatic nerves, seminal vesicles, spinal cord (cervical, thoracic and lumbar), spleen, stomach with forestomach and glandular stomach, testes, thymus, thyroids, trachea, urinary bladder, uterus, vagina, all gross lesions
Statistics:
Clinical examinations:
Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, implantation sites, pups delivered, life pups day x: DUNNETT-test (two-sided).
Male and female mating indices, male and female fertility indices, gestation index, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups: FISHER'S EXACT test (one-sided).
Mating days until day 0 pc, Viability Index: WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment
Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: KRUSKAL-WALLIS test (two-sided), WILCOXON-test (two-sided).

Clinical pathology:
Blood parameters: bidirectional changes: KRUSKAL-WALLIS test, WILCOXON-test (two-sided); unidirectional changes: WILCOXON-test (one-sided).
Urinalysis parameters (apart from urine color and turbidity): WILCOXON-test (one-sided).
Urine pH, volume, specific gravity, color and turbidity: KRUSKAL-WALLIS test, WILCOXON-test (two-sided)

Pathology: weight parameters: KRUSKAL-WALLIS test (two-sided), WILCOXON test (two-sided)

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS, DETAILED CLINICAL OBSERVATIONS AND MORTALITY
Almost all animals of both sexes in test group 3 (1000 mg/kg bw/d) showed slight salivation within 2 hours after the test substance administration on several days of the study. In male animals, salivation was observed beginning on pre-mating day 12 (in-life day 12) as well as during the mating period and the post-mating period. In female animals, salivation started on pre-mating day 12 (in-life day 12) and was observed during the mating, gestation and lactation periods. In test group 2 (300 mg/kg bw/d) one male animal showed slight salivation within 2 hours after the test substance administration on several days of the study, beginning on day 1 of mating phase (study day 14) to day 7 of post-mating phase (study day 21). Two female animals of test group 1 (100 mg/kg bw/d) showed salivation within 2 hours after the test substance administration on several days during the lactation phase (study days 40 to 58). From the temporary, short appearance immediately after dosing it could be assumed that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse.

One female animal of test group 2 (300 mg/kg bw/d) showed reduced nutritional condition and piloerection on lactation days 4 to 6 (study days 45 to 47). The finding was assessed as being incidental but caused by local pathological findings in the glandular stomach.

One female animal of test group 0 (control) showed palpable masses at both mammary lines >1.5 cm on lactation days 6 to 9 (study days 46 to 49). Later, the same animal showed encrusted palpable masses at the left mammary line (>3cm) and an additional palpable mass at the right mammary line (<1.5 cm) from study days 50 to 52. The same animal showed a poor general condition and piloerection from lactation day 6 (study day 46) onwards. This animal was sacrificed in a moribund condition on day 12 of the lactation period (study day 52). One female animal of test group 3 (1000 mg/kg bw/d) showed a dorsal skin lesion from lactation day 8 onwards (study day 48). The findings in these 2 animals were assessed as being spontaneous in nature.

FUNCTIONAL OBSERVATION BATTERY
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.
The following examinations were performed during FOB and assessed individually:
Home cage observations: No test substance-related effects were observed.
Open field observations: One female animal of test group 3 (1000 mg/kg bw/d) showed a dorsal skin lesion. The finding was assessed as being spontaneous in nature and not related to treatment.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative parameters: No test substance-related effects were observed.

MOTOR ACTIVITY MEASUREMENT
Regarding the overall motor activity as well as the single intervals, no test substance-related deviations were noted for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d). Single interval No. 4 in female animals of test group 1 (100 mg/kg bw/d) was significantly lower. This deviation to the control values was regarded as being incidental and not related to treatment as the overall activity was not significantly altered and no dose response-relationship was observed.

BODY WEIGHT
The body weight change was significantly lower in male animals of test group 3 (1000 mg/kg bw/d) between study days 7 to 13 (-48.2%). As no significant differences were observed in mean body weight or during other study periods than between study days 7 to 13 the finding was assessed as being spontaneous in nature and not related to treatment.

FOOD CONSUMPTION
No test substance-related changes with regard to food consumption were observed in most animals of test groups 0-3.
However, one female animal of test group 2 (300 mg/kg bw/d) and of test group 3 (1000 mg/kg bw/d) showed severely reduced food consumption during the lactation period. For the animal of test group 2, the finding was assessed as being incidental but caused by local pathological findings in the glandular stomach. For the animal of test group 3 the finding was assessed as being treatment-related and caused by local pathological findings in the forestomach.

WATER CONSUMPTION
No test substance-related changes with regard to water consumption were observed.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.
In male animals of test group 3 (1000 mg/kg bw/d), relative large unstained cell (LUC) counts were increased. This was the only differential cell count fraction which was increased. Absolute LUC counts were not significantly increased and no alteration of the cell counts occurred in female animals. Therefore, this change was regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.
In male animals of test group 3 (1000 mg/kg bw/d) the urine pH value was decreased. This altered parameter could not be related to any pathophysiological finding and, therefore, it was regarded as maybe treatment-related but not adverse.

ORGAN WEIGHTS
Absolute weights
When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly increased: kidneys (females, test group 2 and 3 - 300 and 1000 mg/kg bw/d). Following organ weights were significantly decreased: adrenal glands (males, test group 2 - 300 mg/kg bw/d) and brain (female, test group 1 - 100 mg/kg bw/d). All other mean absolute weight parameters did not show significant differences when compared to test group 0 (control).

Relative organ weights
When compared to the control group 0 (set to 100%), the mean relative weights of the liver and kidneys were significantly increased in all test groups.
All other mean relative weight parameters in females and all mean relative weight parameters in male animals did not show significant differences when compared to the control group 0. The mean absolute or relative kidney and liver weights in females of all treatment groups were within the range of historical control data, whereas in control females the kidney and liver weights were clearly below the historical data. Therefore, the significantly increase in liver and kidney weights in treated females was considered to be incidental. Because there was no dose-response relationship, the significantly decreased mean absolute adrenal weight in males of test group 2 (300 mg/kg bw/d) as well as the decreased mean absolute brain weight in females of test group 1 (100 mg/kg bw/d) were considered to be incidental.

GROSS PATHOLOGY
One female animal of test group 3 (1000 mg/kg bw/d) showed few erosions/ ulcers (diameter 4mm) in the forestomach that were assessed as being related to treatment.
Few erosions/ ulcers in the glandular stomach were noted in one female of test group 2 (300 mg/kg bw/d). Because few erosions/ ulcers occurred also in the control group in one male animal, the occurrence of erosions in the glandular stomach was considered to be incidental and not related to treatment.
All other findings were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility:
All female animals were pregnant.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver: The number of males with peripheral fatty change was increased in test group 3 (1000 mg/kg bw/d).
For the increased number of males with minimal peripheral fatty change in test group 3 (1000 mg/kg bw/d), a treatment-related effect could not be ruled out. The occurrence of peripheral fatty change in females did not show dose-response relationship and was therefore considered to be incidental. The macroscopically diagnosed erosions/ ulcers in the forestomach of one female animal of test group 3 (1000 mg/kg bw/d) correlated with a moderate focal squamous cell hyperplasia. Histopathologically the finding was interpreted as reactive rim surrounding the erosions/ ulcers. The erosions/ ulcers in the glandular stomach of one female animal of test group 2 (300 mg/kg bw/d) and of the one control male animal was confirmed histopathologically. The one control female animal that was sacrificed moribund showed a massive necrotizing, abscess-forming inflammation in the region of the mammary gland. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were regarded to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were observed.
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No local effects were observed in males.
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on findings in the forestomach noted at 1000 mg/kg bw/day in a single female rat.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Stability analyses

The stability of the test substance in corn oil was demonstrated over a period of 7 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.

Homogeneity control analyses

Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that Delta-Valerolactone was distributed homogeneously in corn oil.

Concentration control analyses

The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of 90-110% of the nominal concentrations. Taken together, the results demonstrate the correctness of the concentrations of Delta-Valerolactone.

Applicant's summary and conclusion