Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD 422 study conducted on the read-across substance, delta-valerolactone, the No Observed Adverse Effect Level (NOAEL) for reproduction and fertility was 1000 mg /kg bw/d (K, rel. 1).

Link to relevant study records

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Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
other: Adaptation according to Section 1.2 of Annex XI
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Justification for type of information:
Adaptation according to Regulation (EC) No 1907/2006 to Section 1 of Annex XI for the Developmental Toxicity Study-Second Species (for more details, please refer to "Attached justification"):
The potential for repeat dose, reproductive and developmental toxicity has been thoroughly investigated in several studies performed on the substance or on analogue substances. The results of these studies indicate that the substance is non-toxic to rats via the oral dose route and that there is no evidence of reproductive or developmental toxicity, even when tested to the limit dose level of 1000 mg/kg bw/d. It is considered that all of the information requirements (foci of examination) of the basic design EOGRT study have been addressed by the existing data.
Based on these study results it is concluded that no further information, of value to evaluate the reproductive and developmental toxicity of the substance, would be gained by the performance of an EOGRT. The existing study data are also considered to be adequate for hazard classification and risk assessment purposes. The ECHA R.7a guidance indicates that “Elements of a weight of evidence adaptation approach according to this adaptation rule for reproductive toxicity could be available from experimental studies addressing reproductive toxicity endpoints…”. The ECHA R.7a guidance also includes a starting assumption that substances with low toxicological activity may be less likely to be reproductive toxicants. The various elements of a WoE argument in terms of existing experimental studies addressing reproductive toxicity endpoints are available and the substance has extremely low toxicity (NOAEL ≥1000 mg/kg bw/d in all studies). In conclusion, it is proposed to waive this standard information requirement on the basis of a WoE approach, according to Annex XI, Section 1.2 and the scientific considerations described above. Metabolism differences between the rat and rabbit have been considered and concluded to have no impact on the reproductive toxicity potential of the substance. This adaptation achieves one of the primary aims of the REACH regulation, to avoid testing in vertebrate animals wherever possible.
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Justification for study design:
Adaptation according to Section 1.2 of Annex XI
Remarks on result:
other: Adaptation according to Section 1.2 of Annex XI
Remarks on result:
other: Adaptation according to Section 1.2 of Annex XI
Remarks on result:
other: Adaptation according to Section 1.2 of Annex XI
Reproductive effects observed:
no
Endpoint:
screening for reproductive / developmental toxicity
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.
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
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
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:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed in almost all animals in the top dose group and in some animals in the other groups. The effect was related to the test substance but assessed as being non-adverse.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, non-treatment-related
Behaviour (functional findings):
effects observed, non-treatment-related
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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
Remarks:
Toxicity to reproduction
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.
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse substance-related effects
Clinical signs:
effects observed, non-treatment-related
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, non-treatment-related
Gross pathological findings:
effects observed, non-treatment-related
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.

Conclusions:
Under the test conditions, the NOAEL for reproductive performance, fertility and developmental toxicity was set to 1000 mg/kg bw/d.
Executive summary:

In a combined repeated dose toxicity with the reproductive/developmental screening test, Delta-Valerolactone was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; 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). Corn oil served as vehicle. The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 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.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the parents was determined once weekly during premating. For the dams food consumption was determined for gestation days 0-7, 7-14, 14-20 and lactation days 1-4. Body weights of parents were determined once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, females were weighed on gestation days (GD) 0, 7, 14 and 20, on the day of parturition (postnatal day [PND] 0) and on PND 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings.

Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group.

Clinicochemical and hematological examinations as well as urinalyses were also performed towards the end of the administration period in 5 animals per sex and test group. All parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The oral administration by gavage of Delta-Valerolactone revealed local pathological findings in the forestomach of one female Wistar rat at a dose level of 1000 mg/kg bw/d. This finding was related to the corrosive potential of the test substance to mucosal membranes. No local pathological findings in the forestomach were observed in female Wistar rats at 300 mg/kg bw/d and male Wistar rats up to a dose level of 1000 mg/kg bw/d.

Signs of systemic toxicity were observed in one female parental animal of test group 3 (1000 mg/kg bw/d) towards the end of the lactation period as indicated by reduced food consumption and secondary effects of maternal toxicity on the litter. The findings were assessed as being related to local pathological findings in the forestomach.

Thus, the NOAEL for reproductive performance, fertility and developmental toxicity was set to 1000 mg/kg bw/d in male and female Wistar rats.

Therefore, the test substance is not classified for Developmental or Reproductive Toxicity according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP).

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
Quality of whole database:
The available information as a whole meets the tonnage driven data requirement of REACH. Moreover, reliability and consistency are observed across the different studies.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No reproduction toxicity study was located on γ-Undecalactone while a reliable study is available on delta-valerolactone, a lactone considered adequate for read-across purpose(see § "Toxicokinetics").

Hence, in a combined repeated dose toxicity with the reproductive/developmental screening test, Delta-Valerolactone was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0, 100, 300 and 1000 mg/kg bw/d. Corn oil served as vehicle. The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 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.

 

The oral administration by gavage of Delta-Valerolactone revealed local pathological findings in the forestomach of one female Wistar rat at a dose level of 1000 mg/kg bw/d. This finding was related to the corrosive potential of the test substance to mucosal membranes. No local pathological findings in the forestomach were observed in female Wistar rats at 300 mg/kg bw/d and male Wistar rats up to a dose level of 1000 mg/kg bw/d.

Signs of systemic toxicity were observed in one female parental animal of test group 3 (1000 mg/kg bw/d) towards the end of the lactation period as indicated by reduced food consumption and secondary effects of maternal toxicity on the litter. The findings were assessed as being related to local pathological findings in the forestomach.

Thus, the NOAEL for reproductive performance, fertility and developmental toxicity was set to 1000 mg/kg bw/d in male and female Wistar rats.

Effects on developmental toxicity

Description of key information

In an OECD 414 study conducted on the read-across substance, ɣ-Caprolactone, the No Observed Adverse Effect Level (NOAEL) for developmental toxicity was 1000 mg /kg bw/d (K, rel. 2).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 26, 2002 to June 14, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other:
Remarks:
OECD 414 guideline study in compliance with GLP. γ-Caprolactone, as a linear saturated 4-hydroxycarboxylic acid derived-lactones, is considered adequate for read-across purpose (see §"Toxicokinetics").
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Caroline
- Age at study initiation: approximately 12 weeks old whenn paired fro breeding
- Weight at study initiation: 237 to 239 g on Day 0 of gestation
- Housing: upon arrival and until paring, individually housed in clean, wire-mesh cages suspended above cage-board. The rats were paired for mating in the home cage of the males. Following positive identification of mating, the femles were returned to an individual suspended wire-mesh cage, nesting material was not required as the females were euthanized prior to the data of expected parturition.
- Diet (e.g. ad libitum): ad libitum, PMI Nutrition International, Inc. Certified Rodent LabDiet® 5002 (analysis certified)
- Water (e.g. ad libitum): ad libitum, reverse-osmosis-treated (on-site) municipal water (analysis certified)
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: March 26, 2002 To: May 3, 2002
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deioniszed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The 1000 mg/kg bw/d test article formulation was a weight/volume (test article/vehicle) mixture. For this group, the appropriate amount of the test article was weighted into a tared weighting vessel. Approximately 60 % of the vehicle was added to a calibrated storage container, and a deep vortex was created with a magnetic stir bar. The test article was slowly transferred quantitatively into the rapidly stirring vehicle and stirred until dissolved (approximately one minute). The remaining amount of vehicle was added to bring the volume to the calibration mark. The formulation was stirred until uniform and throughout use, using a magnetic stirrer. The mixture was used as a stock formulation to prepare the 100 and 300mg/kg bw/d group dosing formulations. For these groups, the appropriate amount of the stock formulation was added to achieve the total volume for each group. The dosing formulations were stored refrigerated, protected from light. The test article formulations were prepared approximately weekly (as single formulations for each dose level), then separated into aliquots for daily dispensation. All test article formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity, re-suspension homogeneity and stability of the test formulations at a similar range of concentrations as used in this study were established in a companion study (Repeated dose toxicity: oral, 28d, Kirkpatrick).
Prior to the initiation of dose administration (April 2, 2002), samples (10 mL each) for homogeneity determination were collected from the top, middle and bottom strata of the all dosing formulations, including the control group. In addition, duplicate samples (10 mL each) for stability determinations were collected from the top and bottom strata of these same dosing suspensions; one set was stored refrigerated for 10 days and one set was stored at room temperature for 10 days. Both sets were protected from light. Samples (10 mL each) for concentration analysis were collected from the first, second and third weekly preparations from each dosing formulation (including the control group.
All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, Inc. The test article formulations were found to be homogenous (the RSD for the overall mean concentration was ≤ 10 % at a concentration that is within the acceptable limits), contain the amount of test article specified in the protocol (analyzed concentrations were within ± 10 % of the target dose concentrations) and stable for at least 10 days when stored refrigerated or at room temperature (the mean concentrations were no less that 90 % of the time zero concentrations).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From GD6 through GD19
Frequency of treatment:
Once daily
Duration of test:
14 days
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: selected based on the results of previous studies and were provided by the sponsor after consultation with the study director.
- Rationale for animal assignment (if not random):
- Other:
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily from day 0 through 20 of gestation. Animals were also observed for signs of toxicity approximately one hour following dose administration.

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0 and 6-20 (daily)
Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-20, 6-20 and 0-20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual food consumption was recorded on gestation day 0 and 6-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding bpdy weigh change intervals . On the occasions when food intake could not be measured for one of the days in a given interval (due to a weighting error, food spillage, obvious erroneous value, etc.), values were calculated using the appropriate number of days for that interval.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No, not a drinking water study

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross observations of organs, maternal tissues were preserved
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 of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Visceral examination: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
- Soft tissue examinations (head): Yes: half per litter
Statistics:
All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for a minimum significance levels of 5 %, comparing each test article-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Due to the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ by ± 1 in the last significant figure.
Mean maternal body weights (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, number of corpora lutea, implantations sites and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistical significant (p < 0.05) intergroup variance, Dunnett’s test was used to compare the test article-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution) were subjected to the Kruskall-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p < 0.05) intergroup variance, the Mann-Whitney U-test was used to compare the test article-related groups to the control group. Mean litter proportions (percent per litter) or total fetal malformations and developmental variations (external, visceral, skeletal and combined) and of each particular external, visceral and skeletal malformation or variation were analyzed by the Kruskall-Wallis nonparametric ANOVA test followed by the Mann-Whitney U-test (if appropriate) as described above.
Indices:
- Postimplantation loss/litter= (No. dead fetuses, resorptions (Early/Late)/Group) / No. Gravid Females/Group.
- Summation per group (%) = Sum of postimplantation loss/litter (%) / No. litters/group.
- Postimplantation loss/litter (%) = (No. dead fetuses, resorptions (Early/Late)/litter) / No. implantation sites/litter * 100
- Viable fetuses affected/litter (%) = (No. viable fetused affected/litter) / No. viable fetuses/litter * 100
Historical control data:
WIL Developmental historical control data [Crl:CD(SD)IGS BR Rats] is incuded in Appendix D of the study report. External, visceral and skeletal malformations and visceral and skeletal deviations were reported for 27 studies (635 fetuses)
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
MATERNAL CLINICAL OBSERVATIONS AND SURVIVAL
All animals survived to the scheduled necropsy. No test article-related findings were observed. Clinical findings, including hair loss or various body surfaces, yellow staining on the ventral abdomen and/or red material around the mouth or nose, were present prior to the initiation of dose administration, occurred infrequently, were noted similarly in the control group and/or did not occur in a dose-related manner.

MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
No test article-related effects on mean body weights, body weight gains, net body weight gains or gravid uterine weights were observed.

MATERNAL FOOD CONSUMPTION
Food consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test article administration. The only statistically significant (p < 0.05 or p < 0.01) differences from the control group were slight decreases in the 300 mg/kg bw/day group (g/kg/day only) during gestation days 17-19. The g/animal/day value in the 1000 mg/kg bw/day group was similar to that in the control group; therefore, the transient reduction in food consumed relative to body weight was not considered to be adverse. A reduction in the g/animal/day value similar to that observed in the 300 mg/kg /day group during gestation days 17-18 was not observed in the 1000 mg/kg bw/day group. Therefore, this transient decrease was not attributed to the test article.

MATERNAL NECROPSY DATA
There were no test article-related internal findings at the scheduled necropsy on GD20. One female in the 100 mg/kg bw/day group had cyst in the renal cortex and medulla (bilateral) and a rough surface of the liver. Another female in this same group had calculi in the urinary bladder, distended ureters and dilated renal pelves. In the 1000 mg/kg bw/day group, one female had dark red areas in the lungs. No other internal findings were observed.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: statistically significant reduced mean fetal body weight

Details on embryotoxic / teratogenic effects:
GD20 LAPAROHYSTERECTOMY DATA
Mean fetal body weights (male, female and combined) in the 1000 mg/kg bw/day group (3.5, 3.3 and 3.4 g, respectively) were reduced (statistically significant at p <0.05 or p < 0.01) compared to the control group value (3.7, 3.5 and 3.6 g, respectively). The values in the 1000 mg/kg bw/day group were equal to the minimum values in the WIL historical control data. The reduction in mean fetal body weight was attributed to the test article. Other intrauterine parameters, including mean viable litter size, fetal sex ratios and mean litter proportions of pre- and postimplantation loss, in this group were unaffected by test article administration.
Intrauterine growth and survival in the 100 and 300 mg/kg bw/day groups were unaffected by test article administration. The mean numbers of corpora lutea and implantation sites were similar in all groups.

FETAL MORPHOLOGICAL DATA
The number of fetuses (litters) available for morphological evaluation were 364(24), 376(24), 365(23) and 393(25) in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Malformations (visceral and skeletal) were observed in 3(3) fetuses (litters) in the 300 mg/kg bw/day group and not treatment relationship as described below.

EXTERNAL MALFORMATIONS AND VARIATIONS
One fetus in the 300 mg/kg bw/day group had a meningocele (a portion of the meninges protruded through an opening of the cranium). No other external malformations were observed.
No external developmental variations were observed.

VISCERAL MALFORMATIONS AND VARIATIONS
One fetus in the 300 mg/kg bw/day group had a malpositioned descending aorta (the aorta coursed laterally to the left adrenal gland, between the adrenal and the left kidney, and resumed the normal position prior to the branching of the iliac artery). No other soft tissue malformations were observed.
Soft tissue developmental variations in the test article-treated groups included distended ureters, major blood vessel variations (the right carotid and right subclavian arteries arose independently from the aortic arch with no brachiocephalic trunk or a retroesophageal right subclavian artery, which joined the aortic arch adjacent to the right carotid artery, with no brachiocephalic trunk), an accessory spleen and a small spleen. These findings were observed in single fetuses or did not occur in a dose-related manner. No relationship to treatment was evident.

SKELETAL MALFORMATIONS AND VARIATIONS
One fetus in the 300 mg/kg bw/day group had a vertebral centra anormality (the right half of lumbar centrum no. 2 was absent, and the right half of lumbar centrum no. 1 was malpositioned). No other skeletal malformations were observed.
Skeletal developmental variations occurred in all dose groups, including the control group, and consisted primarily of unossified sternebra(e) nos. 5 and/or 6, 14th rudimentary ribs, 7th cervical ribs, cervical centrum no. 1 ossified and unossified hyoid. A statistically significant (p < 0.05) increase in the mean litter proportion (% per litter) of 7th cervical ribs was observed in the 100 mg/kg bw/day group compared to the control group value. Similar increases were not observed in the 300 and 1000 mg/kg bw/day groups; therefore this increase was not attributed to test article. Other skeletal variants observed on the test article-treated groups occurred infrequently, were within the range of the WIL historical control data or did not occur in a dose-related manner. No relationship to treatment was evident.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
not specified
Developmental effects observed:
not specified

none

Conclusions:
Under the test conditions, the NOAEL for maternal toxicity was 1000 mg /kg bw/d. Due to slightly reduced foetal mean body weight in the high dose group compared to control, the NOAEL for developmental toxicity was 1000 mg/kg bw/day.
Executive summary:

In a developmental toxicity study conducted according to the OECD guideline No. 414 and in compliance with GLP, γ-Caprolactone diluted in deionized water was administered to 25 females Crl:CD®(SD)IGS BR rats/dose by gavage at dose levels of 0, 100, 300 or 1000 mg/kg bw/day from days 6 through 19 of gestation (dose volume = 10 mL/kg bw).

 

All animals were observed twice daily for appearance and behavior. Clinical observations, bodyweight and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uteri and ovaries were examined, and the number of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighted, sexed and examined for external, visceral and skeletal malformations and developmental variation.

 

All animals survived to the scheduled necropsy on GD20. There were no test article-related maternal clinical observations or effects on mean body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights or food consumption at any dose level. There were no test article-related internal findings at the scheduled necropsy. The mean fetal sex ratio and mean numbers and/or litter proportions of viable fetuses and pre- and postimplantation losses in the 1000 mg/kg bw/day group were not affected by test article administration. Intrauterine growth and survival were unaffected in the 100 and 300 mg/kg bw/day groups. No test article-related fetal malformations or developmental variations were noted at any dose level in this study.

Test article-related effects noted in the 1000 mg/kg bw/day group consisted of statistically significant reduced mean fetal body weight (5.6 % lower than the control group value for combined fetal weight). This slight decrease was not considered of toxicological concern. No other indicators of developmental toxicity were noted.

Based on the results of this study, the dose level of 1000 mg/kg bw/day was considered to be the NOAEL for maternal toxicity and the dose level of 1000 mg/kg bw/day was considered to the NOAEL for developmental toxicity.

Under the test conditions, γ-Caprolactone is not classified according to Regulation (EC) No.1272/2008 (CLP) criteria.

This study is acceptable and satisfies the requirement for developmental toxicity endpoint.

Endpoint:
developmental toxicity
Remarks:
second species
Type of information:
other: Adaptation according to Section 1.2 of Annex XI
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Justification for type of information:
Adaptation according to Regulation (EC) No 1907/2006 to Section 1 of Annex XI for the Developmental Toxicity Study-Second Species (for more details, please refer to "Attached justification"):
According to REACH Regulation Annex XI, Section 1.2. Weight of evidence:
‘There may be sufficient weight of evidence from several independent sources of information leading to the assumption/conclusion that a substance has or has not a particular dangerous property, while the information from each single source alone is regarded insufficient to support this notion……………
………….Where sufficient weight of evidence for the presence or absence of a particular dangerous property is available:
- further testing on vertebrate animals for that property shall be omitted……..’
The absence of developmental and reproductive toxicity effects at any dose in two high quality relevant studies performed in rats on analogue substances, indicates that there is no concern for developmental toxicity for this substance. Furthermore, published data indicates that for substances of low concern for developmental toxicity there is no value in the performance of a study in a second species. On this basis, and taking account of REACH Annex XI, Section 1.2, the several independent sources of information provide sufficient evidence to conclude that the substance does not have the potential for developmental toxicity in a second species (rabbit). The ECHA R.7a guidance indicates that “Elements of a weight of evidence adaptation approach according to this adaptation rule for reproductive toxicity could be available from experimental studies addressing reproductive toxicity endpoints…”.
The ECHA R.7a guidance also includes a starting assumption that substances with low toxicological activity may be less likely to be reproductive toxicants. The various elements of a WoE argument in terms of existing experimental studies addressing reproductive toxicity endpoints are available and the substance has extremely low toxicity (NOAEL ≥1000 mg/kg bw/d in all studies performed on the substance or on analogue substances). In conclusion, it is proposed to waive this standard information requirement on the basis of a WoE approach, according to Annex XI, Section 1.2 and the scientific considerations described above. Metabolism differences between the rat and rabbit have been considered and concluded to have no impact on the reproductive toxicity potential of the substance. This adaptation achieves one of the primary aims of the REACH regulation, to avoid testing in vertebrate animals wherever possible.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Species:
rabbit
Remarks on result:
other: Adaptation according to Annex XI, Section 1.2
Remarks on result:
other: Adaptation according to Annex XI, Section 1.2
Developmental effects observed:
no
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
Quality of whole database:
The available information as a whole meets the tonnage driven data requirement of REACH. Moreover, reliability and consistency are observed across the different studies.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

No developmental toxicity study was located on γ-Undecalactone while a reliable study is available on γ-Caprolactone, a linear saturated 4-hydroxycarboxylic acid derived-lactones considered adequate for read-across purpose(see § "Toxicokinetics").

Hence, in a developmental toxicity study conducted according to the OECD guideline No. 414 and in compliance with GLP, γ-Caprolactone diluted in deionized water was administered to 25 females Crl:CD®(SD)IGS BR rats/dose by gavage at dose levels of 0, 100, 300 or 1000 mg/kg bw/day from days 6 through 19 of gestation.

 

All animals survived to the scheduled necropsy on GD20. There were no test article-related maternal clinical observations or effects on mean body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights or food consumption at any dose level. There were no test article-related internal findings at the scheduled necropsy. The mean fetal sex ratio and mean numbers and/or litter proportions of viable fetuses and pre- and postimplantation losses in the 1000 mg/kg bw/day group were not affected by test article administration. Intrauterine growth and survival were unaffected in the 100 and 300 mg/kg bw/day groups. No test article-related fetal malformations or developmental variations were noted at any dose level in this study.

Test article-related effects noted in the 1000 mg/kg bw/day group consisted of statistically significant reduced mean fetal body weight (5.6 % lower than the control group value for combined fetal weight). This slight decrease was not considered of toxicological concern. No other indicators of developmental toxicity were noted. No other indicators of developmental toxicity were noted.

Based on the results of this study, the dose level of 1000 mg/kg bw/day was considered to be the NOAEL for both maternal toxicity and developmental toxicity.

Justification for classification or non-classification

Harmonized classification:

ɣ-Undecalactone has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed for reproductive or developmental toxicity according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and the GHS.