Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
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.
Reason / purpose:
reference to same study
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
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
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on 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)
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight, until there was evidence of copulation, or a maximum period of 14 days
- Further matings after two unsuccessful attempts: no
- Proof of pregnancy: sperm in vaginal smear. The day on which sperm was detected was reffered to as gestation day (GD) 0 and the following day as GD 1.
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
Details on study schedule:
- Age at mating of the mated animals in the study: 14 - 15 weeks old
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
Parental animals: Observations and examinations:
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.
Oestrous cyclicity (parental animals):
Not investigated.
Sperm parameters (parental animals):
Not investigated.
Litter observations:
PUP NUMBER AND STATUS AT DELIVERY
All pups delivered from the F0 parents were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn pups in each litter. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.

SEX RATIO
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.

The sex ratio was calculated at day 0 and day 4 after birth according to the following formula:
Sex ratio = (number of live male or female pups on day 0/4 /number of live male and female pups on day 0/4) x 100

PUP CLINICAL OBSERVATIONS
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

PUP BODY WEIGHT DATA
The pups were weighed on the day after birth (PND 1) and on PND 4.
Pups body weight change was calculated from these results.
The individual weights were always determined at about the same time of the day (in the morning).
“Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
Postmortem examinations (parental animals):
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
Postmortem examinations (offspring):
GROSS NECROPSY
- All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Not performed.
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)
Reproductive indices:
Male mating index
Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) x 100
* defined by a female with vaginal sperm or with implants in utero

Male fertility index
Male fertility index (%) = (number of males proving their fertility*/number of males placed with females) x 100
* defined by a female with implants in utero

Female mating index
Female mating index (%) = (number of females mated*/number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index
Female fertility index (%) = (number of females pregnant*/number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index
Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100
* defined as the number of females with implants in utero

Live birth index
Live birth index (%) = (number of liveborn pups at birth/total number of pups born) x 100
Offspring viability indices:
Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated according to the following formula:
Viability index (%) = (number of live pups on day 4 after birth/number of live pups on the day of birth) x 100

CLINICAL SIGNS, DETAILED CLINICAL OBSERVATIONS AND MORTALITY (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
No test substance-related changes with regard to water consumption were observed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Male reproduction data:
For all F0 parental males, which were placed with females to generate F1 pups, mating was confirmed. Thus, the male mating index was 100% in all test groups (0, 100, 300 and 1000 mg/kg bw/d).
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Thus, the male fertility index was 100% in all test groups.

Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter was 100% for all test groups (0, 100, 300 and 1000 mg/kg bw/d). The mean duration until sperm was detected (GD 0) was 2.4, 2.3, 2.8 and 3.8 days in test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d).
All rats delivered pups. The female fertility index was 100% for all test groups.
The mean duration of gestation was between 22.1 and 22.3 days and did not show significant differences between the test groups.
The gestation index reached 100% in all test groups including the control group.
The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% in all test groups.

HAEMATOLOGY (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
No treatment-related changes among clinical chemistry parameters were observed.

URINALYSIS (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 significant 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 (PARENTAL ANIMALS)
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.

HISTOPATHOLOGY(PARENTAL ANIMALS) : 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.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive effects were noted.
NUMBER AND STATUS AT DELIVERY (OFFSPRING)
The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were not affected by the test substance.

VIABILITY/MORTALITY (OFFSPRING)
The viability index as indicator for pup mortality between PND 0 and 4 was 100% for test groups 0 and 1 (0 and 100 mg/kg bw/d). The viability index was slightly decreased down to 93.8% in test group 2 (300 mg/kg bw/d) and 99.3% in test group 3 (1000 mg/kg bw/d). The decrease was related to each 1 litter in these test groups, i.e. the litters of one parental female of test group 2 and one of test group 3. The parental females showed gross lesions in the glandular stomach (one female of test group 2 - 300 mg/kg bw/day) and the forestomach (one female of test group 3 - 1000 mg/kg bw/day) what was most likely the reason for stressed behavior. Therefore, the pups were either not properly nursed and found dead (the female of test group 2 - 300 mg/kg bw/day) or even cannibalized (the female of test group 3 - 1000 mg/kg bw/day). The decreased pup viability in both litters was assessed as being secondary to the impaired maternal condition. A direct effect of the test substance on the pups was not assumed.
Even though, the decreased viability index was within the historical control data and reflected the normal range of biological variation inherent in the strain used in this study.

SEX RATIO (OFFSPRING)
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups. All deviations observed for test groups 1-3 compared to the controls reflected the normal range typical for this rat strain.

CLINICAL OBSERVATIONS (OFFSPRING)
Reduced nutritional condition was observed in 6 male and 7 female pups in test group 2 (300 mg/kg bw/d) and 7 male and 7 female pups in test group 3 (1000 mg/kg bw/d). Again, this type of finding was related to each 1 litter in these test groups, i.e. the litters of same parental female of test group 2 and of test group 3. The parental females showed gross lesions in the glandular stomach (the female of test group 2 - 300 mg/kg bw/day) and the forestomach (the female of test group 3 - 1000 mg/kg bw/day) what was most likely the reason for stressed behavior. Therefore, the pups were not properly nursed. The occurrence of reduced nutritional status in both litters was assessed as being secondary to the impaired maternal condition. A direct effect of the test substance on the pups was not assumed.

BODY WEIGHT (OFFSPRING)
The mean body weight of female pups in test group 2 (300 mg/kg bw/d) was significantly lower on PND 1 (-13.8%) as well as on PND 4 (-17.4%) as compared with the control group. On PND 4 the body weight of males + females was significantly lower (-15.8%). These findings were assessed as being related to the litter of the female of test group 2 (300 mg/kg bw/d) as a higher amount of runts was found, i.e. 6 male and 7 female animals. This dam itself also showed reduced nutritional condition and piloerection on lactation days 4 to 6 (study days 45 to 47), what was most probably be related to gross lesions in the glandular stomach. Given that, it was assumed that a proper nursing of the pups was not given what in turn influenced the weight development.
Runts were also observed in individual litters of all test groups. On PND 1, 1 male runt was seen in the litter of one parental animal and 1 female runt in the litter of another parental animal of test group 0 (control). Also, 1 male runt was seen in the litter of one parental animal of test group 1 (100 mg/kg bw/d). In test group 2 (300 mg/kg bw/d), 1 male and 1 female runt of one parental animal, 2 female runts of another parental animal and 1 female runt of a third parental animal were determined. In test group 3 (1000 mg/kg bw/d), 2 male runts of one parental animal, 1 male runt of another parental animal as well as 1 male and 2 female runts of a third parental animal were determined. These findings were assessed as being incidental.

NECROPSY OBSERVATIONS (OFFSPRING)
An empty stomach was seen in nearly the complete litter of the one parental animal of test group 2 (300 mg/kg bw/d), i.e. 6 male and 6 female pups. This parental animal was most likely not able to nurse the pups properly. The same was true for one parental animal of test group 3 (1000 mg/kg bw/d), which had 3 male and 6 female pups with empty stomachs.
Cloudy discolored livers were seen in individual pups of test group 2 (300 mg/kg bw/d), i.e. 1 male pup in the litter of one parental animal, 2 female pups in the litter of another parental animal and 1 female pup of a third parental animal . In test group 3 (1000 mg/kg bw/d) 1 male and 1 female pup in the litter of one parental animal showed the same finding. A hydroureter and fused kidney was observed in 1 female pup in litter of one parental animal of test group 2 (300 mg/kg bw/d). All of these findings were assessed as being incidental and not related to treatment.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental effects were noted.
Reproductive effects observed:
not specified

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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

Toxicity to reproduction: oral in rats

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (BASF SE, 2013) Delta-Valerolactone was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/day (test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). The animals of the control group were treated in the same way with the vehicle only (corn oil). Fourteen days after the beginning of treatment, males and females from the same test group were mated. The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 3 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice. Clinical and detailed clinical signs, food and water consumption as well as body weight development and gain were determined repeatelly during the study period. Towards study end, clinicochemical and haematological examinations, urinalysis and a functional observation battery incl. motor activity measurements were performed. After sacrifice, parental animals were examined by gross pathology. Selected organs were weighed and a histopathological examination was performed. Pups were sexed and macroscopically examined on PND0. Viability was recorded. They were weighed on PND1 and PND4. Necropsy was performed on PND4. All pups were examined macroscopically for external and visceral findings. Since a detailed study summary and conclusion is already presented in the section on repeated dose toxicity (section 7.5), the results are only briefly presented.

Results:

Number and status at delivery:

The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were not affected by the test substance.

Viability/mortality:

A direct effect of the test substance on the pups was not observed.

Sex ratio:

The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups.

Clinical observations:

The occurrence of reduced nutritional status in two litters was assessed as being secondary to the impaired maternal condition due to local toxicity. A direct effect of the test substance on the pups was not assumed.

Body weight:

All observed findings were assessed as being incidental.

Necropsy observations:

All findings observed were assessed as being incidental and not related to treatment.

Waiver: 2-generation reproduction study

According to REACH regulation Annex IX column 1 additional testing for toxicity to reproduction is not necessary because the available data on repeated dose toxicity (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test) did not indicate adverse effects on reproductive organs, tissues or behaviour.


Short description of key information:
NOAEL systemic parental toxicity: 1000 mg/kg bw/d
NOAEL local parental toxicity: 1000 mg/kg bw/d (males), 300 mg/kg bw/d (females)
NOAEL reproduction/fertility toxicity: 1000 mg/kg bw/d
NOAEL developmental toxicity: 1000 mg/kg bw/d

Justification for selection of Effect on fertility via oral route:
The key study was selected (guideline study conducted in accordance with GLP).

Effects on developmental toxicity

Description of key information
In a prenatal developmental toxicity study in rats according to OECD 414, Delta-Valerolactone was administered once daily by oral gavage from days 6 to 20 post-coitum at doses of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, corn oil, alone. No maternal or developmental toxicity was observed up to the limit dose of 1000 mg/kg bw/day.Thus, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Delta-Valerolactone was established as being at least 1000 mg/kg bw/day.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Feb 2016 - 03 Mar 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(Jan. 2001)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
(May 2008)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
(August 1998)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 10 to 14 weeks
- Weight at study initiation: 203 - 207 g
- Housing: individually in Macrolon plastic cages (MIII type)
- Diet (e.g. ad libitum): ad lib. (SM R/M-Z from SSNIFF)
- Water (e.g. ad libitum): ad lib. (tap water)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): at least 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12h / 12h
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
specific gravity 0.92
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test item (1.107) and vehicle (0.92). No correction was made for the purity/composition of the test item.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations
- Concentration in vehicle: 100, 300, 1000 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (16 February 2016), according to a validated method (Project 512208). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also
determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy:Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug
Duration of treatment / exposure:
From Days 6 to 20 post-coitum, inclusive
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Duration of test:
All animals were sacrificed on Day 21 post-coitum
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected by the Sponsor and based the results of an OECD422 study, in which no systemic toxicity was observed up to 1000 mg/kg bw/day.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative assessment was introduced.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21 post-coitum
- Organs examined: All macroscopic abnormalities (external, thoracic, abdominal) were recorded, collected and fixed in 10% buffered formalin.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number and distribution of implantations: Yes (In case implantations were not macroscopically visible, the uterus was stained using the Salewski technique in order to determine any former implantation sites )
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: sex and weight of each fetus, weight of each placenta (of live fetuses only)
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter, i.e., the fetuses also used for visceral examination]
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
- The Steel-test many-to-one rank test)) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Pre-implantation loss(%) = (number of corpora lutea - number of implantation sites) / number of corpora lutea x100
Post-implantation loss(%) = (number of implantation sites - number of live fetuses) / number of implantation sites x100
Viable fetuses affected/litter(%) = number of viable fetuses affected/litter / number of viable fetuses/litter x100
Details on maternal toxic effects:
Maternal toxic effects: no

Macroscopic examination at necropsy revealed no treatment-related findings.
One female at 300 mg/kg bw/day showed a diaphragmatic hernia of the liver. This was considered as incidental and not treatment related.
A broken right upper incisor was noted for one female (no. 23) at 100 mg/kg bw/day, confirming the clinical sign observed during the in-life phase.

One female at 100 mg/kg bw/day and one female at 1000 mg/kg bw/day were non-pregnant. All pregnant females had litters with viable fetuses.
A relatively high mean value for pre-implantation loss (14.6% per litter) was noted in the control group compared to the treatment groups. As this value was within normal limits and concerned the control females, it is not considered toxicologically significant.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
There were no treatment-related effects on litter size for any group

The male:female ratio was unaffected by treatment up to 1000 mg/kg bw/day

There were no effects on fetal body weights (per sex and combined for both sexes) noted by treatment up to 1000 mg/kg bw/day.
8.4.1. External malformations and variations
There were no treatment related effects on external morphology following treatment up to 1000 mg/kg
bw/day.

The only external malformation observed in this study was noted in a fetus of Group 3. The affected fetus (A063-10) had an omphalocele and since this finding occurred singly, it was considered a chance finding.
External variations were not seen in any group

There were no treatment related effects on visceral morphology following treatment up to 1000 mg/kg bw/day.

Two malformations were revealed at fetal visceral examination. Group 4 fetus A081-10 appeared to have an absent eye and a small eye at serial sectioning of the head. In control fetus A014-03 the aortic arch was on the right rather than on the left side. The single occurrence of an eye malformation at the highest dose level, a finding that was also previously noted among historical control fetuses, was not considered to be related to treatment.
The variation of appendix of the liver showed statistically significantly higher incidences in Groups 2 and 3. Mean litter incidences were 0.0%, 3.9%, 6.4% and 2.4% per litter for Groups, 1, 2, 3 and 4, respectively. However, because the group distribution of this variation occurs also spontaneously and does not denote a dose response, it was not considered to be attributed to treatment.
Other variations that were noted in this study were small supernumerary lobe(s) of the liver, partially undescended thymus horns and dilated and convoluted ureters. These variations occurred at low incidences, in the absence of a dose-related incidence trend and/or were noted in control fetuses only and therefore were not considered to be treatment related.

There were no treatment related effects on skeletal morphology following treatment up to 1000 mg/kg bw/day.
Only one skeletal malformation was observed (Group 2 fetus A042-02 had an interrupted cervical vertebral arch). The variations that were noted occurred in the absence of a dose-related incidence trend or occurred infrequently or were within the historical control range of the rat strain used. Therefore, all skeletal findings were not considered treatment related
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
IMaternal findings
No maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.
Developmental findings
No developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.
In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Delta-Valerolactone was established as being at least 1000 mg/kg bw/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Justification for classification or non-classification

Classification is not warranted according to the criteria of EU Directive 67/548/EEC and Regulation 1272/2008/EC.