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Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Specific details on test material used for the study:
Test Item
gamma-Dodecalactone
Lot Number
050170905
Purity
99.02 %
CAS No
2305-05-7
EINECS-No
218-971-6
Appearance
colourless liquid
Composition
gamma-Dodecalactone
Production Date
2017-09-19
Expiry Date
2018-09-18
Storage
Room temperature (20 ± 5° C)
Test Item Handling and Storage
According to SPPA-00147-BIO, Test and Reference Items
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species
Wistar rats
Source
Velaz, Czech Republic
Number and Sex of Animals
50 males + 62 females
Age
At 8-12 weeks; female animals were non-pregnant and nulliparous
Animal Health
The health condition of animals was examined by a veterinarian
before initiation of the study
Acclimation
The animals were acclimated to the condition identical to the condition during the experiment 5 days prior to the start of treatment. The acclimation was according to standard operation procedures.
Housing Condition
The animals were housed in plastic cages suspended on stainless steel racks in a room equipped with central air-conditioning. The room temperature was within the range of 22 ± 2°C; relative humidity was within the range of 55 ± 10 %. The light regimen was set to a 12-hour light / 12-hour dark cycle The sanitation was performed according to standard operation procedures.
Diet
A laboratory food ssniff (ssniff Spezialdiäten GmbH) was offered in recommended doses each day approximately at the same time. The certificate of analysis was included in the raw data.
Water
The animals were received tap water for human consumption. Supply of drinking water was unlimited. The quality of drinking water is periodical monitored and recorded; certificate of analysis was included in raw data.
Bedding
Lignocel S 3/4, Lufa - ITL GmbH, Germany. The certificate of analysis was included in the raw data.
Animals Identification
Each animal was marked with an ID number. Each cage was affixed with a cage card containing pertinent animal and study information. The animals in cages were marked by a line on the tail with an ink marker.
Number of animals in cage was according period of study:
 Pre-treatment period (14 days) – 5 animals per cage
 Pre-mating (14 days) – 5 animals per cage
 Mating (maximum 14 days) - 1 male/1 female per cage
 Gestation (approximately 22 days) - 1 female per cage
 Post-partum (13 days) - 1 female with offspring per cage
Satellite animals – 5 animals per cage during all the study
Justification for the Choice of Species
The Test Guideline OECD 422 is designed for use with the rat. The rats are the standard experimental rodent of choice and recommended by OECD Guideline and in the international validation program the rat was the only species used for the detection of endocrine disrupters
Route of administration:
oral: gavage
Details on route of administration:
Route
Per oral by gavage
Dose volume was 2.0 mL/kg of body weight and was adjusted according to the weight development of the animals.
Vehicle:
olive oil
Details on oral exposure:
The test item was administered in a single dose by gavage using a metal stomach tube.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of gamma-Dodecalactone in formulations containing test item in concentrations of 25 mg/mL, 150 mg/mL and 375 mg/mL in olive oil was determined by GC method. Method code 6221411 is attached in Appendix 7.
Method was validated on 3 concentrations of application solutions, where specificity, selectivity, sensitivity, linearity, accuracy and repeatability were assessed. The validation was performed in consideration of the current ICH Guideline Q2 (R1) based on the corresponding validation protocol VALR-00864-QC.
Duration of treatment / exposure:
Frequency & Duration
Once per day.
Females were treated during:
 14-day pre-mating,
 14-day mating (maximum)
 22-day gestation (approximately)
 13-day lactation
Males were treated during:
 14-day pre-mating,
 14-day mating (maximum)
The animals designated for post-treatment observation (5 animals per sex in control and high groups, respectively) remained untreated for subsequent 14 days.
Frequency of treatment:
Frequency & Duration
Once per day.
Females were treated during:
 14-day pre-mating,
 14-day mating (maximum)
 22-day gestation (approximately)
 13-day lactation
Males were treated during:
 14-day pre-mating,
 14-day mating (maximum)
The animals designated for post-treatment observation (5 animals per sex in control and high groups, respectively) remained untreated for subsequent 14 days.
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Dosing of both sexes began 2 weeks prior to mating. The females were screened for normal oestrous cycles (in a 2-week pre-treatment period).
Dosing continued in both sexes during the mating period. Males were further dosed after the mating period until total dosing period of 28 days. They were then killed.
Daily dosing of the parental females continued throughout pregnancy up to and including, Day 13 post-partum or the day before sacrifice.
Animals in a satellite group scheduled for follow-up observations were not mated. Recovery period to detect delayed occurrence, persistence of, or recovery from toxic effects in males was 14 days after 28-days treatment period; females were kept at 14 days after the last treatment of the last pregnant female.
Observations and examinations performed and frequency:
Observation
All animals were checked for morbidity or mortality at twice daily. The health condition of the animals, behaviour, reaction of the animals to the applied item, their well-beings were monitored and recorded in raw data.

General Clinical Observations
General clinical observations were performed once a day, 2 hours after dosing. Detailed clinical observations were made in all parental animals prior to the first exposure, and at a week thereafter. The signs as changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity as well as changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies were recorded.

Functional Observations
The functional observations were conducted in five males and five females, randomly selected from each group included:
- Open field test
- Tail flick test
- Grip-strength test
Open field test was made of a dark polyvinyl plastic with dimensions of 60 x 60 cm surrounded by 25 cm high walls. Each session started by placing the rat in the central area of the maze. Test duration was 10 min. The activity was recorded and analysed using ANY-Maze Videotracking Software.
In males the Tail flick test was made shortly before scheduled kill. In females the test was made once during the last week of lactation, shortly before scheduled kill. The tail flick latency was assessed using a tail-flick-meter. A light sensor located under the tail indicated the withdrawal of the tail. The tail was placed on a level surface, a radiant heat was applied to the tail and the latency of the rat to remove its tail from the heat was recorded. A maximum tail-flick latency of 10 sec. was used to minimise tissue damage.
For muscle strength assessment, a grip strength meter was used. To perform the evaluation, the animal was pulled by the tail with regular force. The rat could seize a grid attached to a force transducer, till the animal lost its grip. The test was repeated 3 times per forelimb and the maximum grip force (strength in grams) per trial was included in the statistical analysis.

Laboratory Examinations
Blood samples were collected from fasted animals; haematology and clinical chemistry parameters were analysed preferably at the same day as blood was collected. Plasma samples for determination of coagulation test and thyroid hormone levels were stored according relevant condition for later analysis. Blood collection for haematological investigations was performed from the incision on the tail; the blood collection for clinical chemistry (serum) from retrobulbar venoplex was performed under anaesthesia (Isofluran). Blood collection for coagulation tests (citrate plasma) and thyroid hormone (EDTA plasma) was taken from the heart.
The blood collection, processing of the blood and the determination of its haematological and clinical chemistry parameters were performed according to the standard operation procedures.
Sacrifice and pathology:
All test animals were subjected to gross necropsy. Full, detailed gross necropsy included careful examination of external surface of the body, all orifices, and cranial, thoracic and abdominal cavities and their contents. All gross pathological changes were recorded for each animal.
The pups were euthanized on PND 13 and examined for gross abnormalities with particular attention to the reproduction organs. From all adult males and females and one male and female pup from each litter thyroid glands were preserved on PND 13.
Histopathology
The tissues for histology from all animals were preserved in 10% neutral buffered formalin, testes in Davidson fixative and prepared in the paraffin technical.

Organs of the reproductive system
The number of implantation site
Vaginal smears
The wet weight of testes and epididymides, prostate and seminal vesicles with coagulating glands as a whole of all male adult animals
The ovaries, testes, epididymides, accessory sex organs, and all organs showing macroscopic lesions of all adult animals, were preserved
In addition, for a least five adult males and females, randomly selected from each group
Wet Weight
Histopatology
Liver
all gross lesions
Kidneys
brain (representative regions including cerebrum, cerebellum and pons)
Adrenals
spinal cord
Thymus
eye
Spleen
stomach
Brain
small and large intestines (including Peyer's patches)
Heart
liver
kidneys
adrenals
spleen
heart
thymus
trachea
lungs
gonads (testis and ovaries)
accessory sex organs (uterus and cervix, epididymides, prostate, seminal vesicles plus coagulating glands)
vagina
urinary bladder
lymph nodes
Peripheral nerve (sciatic or tibial)
skeletal muscle and bone, with bone marrow
Dead pups and pups killed at Day 13 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which should be examined for signs of altered development.
Statistics:
Individual data (five males and all pregnant females) of clinical chemistry, haematology, body weight, relative weight of organs, results of tail flick test and grip-strength test and T4 levels obtained in the experiment were assessed applying statistical software StatgraphicsTM Centurion. Kruskal-Wallis statistical procedure of multiple comparison was adopted to test the null hypothesis that the medians among the dose groups (control, low, mid, high) are the same. The p-value of 0.05 was considered as the level of statistical significance. In the case of statistically significant outcome the Kruskal-Wallis was followed by Mann-Whitney W test to determine which medians are different from which other. Basic descriptive statistics (mean, SD) are reported as well.
Reproduction/developmental and locomotor activity data were analysed using STATISTICA 7.0 (Statsoft, Inc. Tulsa, OK) software. The data are represented as mean ± S.E.M (and were analysed by one-way analyses of variance (ANOVA) followed by Dunnet’s post-hoc test for a multiple comparison procedure to compare each of a number of treatments with a single control. The p<0.05 value was considered statistically significant.
Clinical signs:
no effects observed
Description (incidence and severity):
All other males and females at all dosage levels survived to the scheduled necropsy without significant visible clinical signs.
Mortality:
no mortality observed
Description (incidence):
During the study one mortality was recorded. One males of High dose satellite (ID 49) died on Day 19 of the treatment. The cause of death was not being determined precisely, because the animal was found in a state of autolysis.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight of males of all dose groups was accordingly increasing during the study. No significant differences between control and dose groups were observed. The body weight of High dose satellite males was similar in comparison with recovery Control males satellite during the whole study.
The body weight of females of all dose groups was mildly increasing during the study. Statistically significant increase of body weight in females of Low and Mid dose against Control on Day 1 and Day 7 of gestation and Day 1 of lactation was recorded.
The body weight of recovery High dose females was similar in comparison with recovery control females during the whole study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of males of all dose groups was similar to the control males during the whole study. Food consumption of recovery treated males was similar in comparison with control group during the whole application and recovery period.
Food consumption of treated females was similar in comparison with control group during the whole application period. Food consumption of recovery treated females was similar in comparison with recovery control females for the whole study.
Haematological findings:
no effects observed
Description (incidence and severity):
No differences between control and doses group in males were observed. In males of High dose satellite statistically significant increase of lymphocytes (Lym), monocytes (Mon) and granulocytes (Gra) against Control group were registered.
Only statistically significant decrease of prothrombin time (PT) in female of Mid dose was registered. No differences between control and High dose satellite females were noticed.
These sporadic changes had no dose relationship; they were considered to be a result of intra-individual and inter-individual variability for this species or they have only statistical character.
No test item related effects on the haematology parameters were observed in this study. During the study, haematology parameters in both sexes were within or close to the historical control data for this species.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Statistically significant increase concentration of calcium (Cal) and decrease of concentration of inorganic phosphorus (PHOS) in males of High dose were registered. Statistically significant decrease of concentration of PHOS was registered in satellite males of High dose, too.
In female only statistically significant decrease of urea in satellite animals of High dose was noticed.
There were no findings in clinical chemistry parameters which could be definitively attributed to the treatment.
The average values of clinical chemistry parameters in all animals were within or slightly outside the serum historical control data.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No significant changes against normal physiological conditions in males were detected. In the urine of some animals, small amounts of protein, ketones and presence of leukocytes were observed. There are no differences between control and the dose groups. These findings considered to be normal (6). No test item related effect was observed.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Open field test
The locomotor activity was not influenced by the administration of tested item in neither experimental group in both sex compared to Control.

Tail flick Test
Statistically significant difference between the Control and the High dose was observed in satellite males. This significance has probably only statistical character. Other differences observed within the males group were not statistically significant.
In comparison with Control, the reaction time was not statistically significant influenced by the administration of the test item in females.

Grip Strength Test
As to the group of males, females and males satellite, there is not a statistically significant difference between the means of component groups. In female satellite the difference between the means of Control and High dose proved to be statistically significant. This significance has probably only statistical character.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no changes in the relative weights of the thyroid glands in males and females in all test item treatment groups compared with the control group.
Statistically significant decrease relative weight of thymus in Mid and High dose against Control in males were registered. In satellite males statistically significant decrease relative weight of left adrenal and increase of thymus were registered.
These changes were minimal and have only statistical character. No changes in relative weight of organs in females were noticed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All animals were necropsied. In one female (ID 102 High dose) hepatomegaly and splenomegaly was registered; rest animals had organs were without visible pathological findings. No other macroscopically changes were noticed.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathology examinations were performed for a five adult males and females, from Control and High group; reproduction organs were performed on all animals of this groups after preparation of paraffin sections and hematoxylin-eosin staining.
The etiology of follicular cysts (ID 96F/H Ov-623 cyst) remains a mystery, in part due to the difficulty of studying them during their formation (2). Follicular cysts may be associated with oestrogenic compounds or they may develop from pre-ovulating follicles which fail to ovulate (7). Lesion is not related to the toxicological study.
Mucification of cervix (ID54F/C CU-485; ID59F/C CU- 498; ID60F/C CU-502; ID63F/C CU-510; ID98F/H CU-685; ID104F/H CU 736) is a common change in rodents (7). Lesion is not related with administered item in experimental animals.
Cystic dilation of acini (ID10M/C Pr-200, M/C Pr-28, ID39M/H Pr-327) consists of distension of the gland with secretory material. This condition is usually associated with older age (14). Lesion is not related to the toxicological study.
Golden brown pigment in uterus wall (ID60F/C U-503, ID62F/CU-507). Hemosiderin may be secondary to haemorrhage, especially in multiparous females at sites of placentation and some are ubiquitous in aging animals (3). Lesion is not related to the toxicological study.
Dilatation of submucosal glands of trachea (ID1M/C Tra-19; ID3M/C Tra-85) may occur following blockage of excretory ducts secondary to hyperplasia or squamous metaplasia of mucosal epithelium, or secondary to inflammation and may arise spontaneously (13). Lesion is not related to the toxicological study.
In this study, observed changes are considered to be incidental findings or results of experimental manipulation other than administration of the test item. There were no test item - related alterations in the prevalence, severity or histological character of these incidental found lesions.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
urinalysis
Key result
Critical effects observed:
no
Conclusions:
After consideration of the results of this study following conclusions were made regarding the test item gamma-Dodecalactone:
The test item
- did not cause mortality of animals
- had no visible toxic effect on animals
- had no influence on haematological and clinical chemistry parameters
- did not cause changes in urine of males
- had no influence on the locomotor activity, reaction time and grip strength
- did not influenced length of pregnancy, survival of pups, AGD or other variables
- did not cause of macroscopic findings and histopathological changes on the examined organs
No-observed-adverse-effect level (NOAEL)
Based on the reproduction toxicity
• NOAEL for males and females was concluded to be 750 mg/kg
Based on the systemic toxicity
• NOAEL for systemic toxicity in males and females was concluded to be 750 mg/kg
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification