Hexan-4-olide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

Vehicle:

olive oil

Details on exposure:

The test item was administered in a single dose by gavage using a metal stomach tube. Administration volume was 2 mL/kg. For each animal the individual dosing volume was calculated on the basis of the actual body weight.

Details on mating procedure:

Normally, 1:1 (one male to one female) matings were used in this study. The female was placed with the same male of the same group until pregnancy occurs or two weeks have elapsed. Each morning the females were examined for the presence of sperm. Day 0 of pregnancy was defined as the day on which mating evidence is confirmed (sperm is found). In case pairing was unsuccessful, re-mating of females with proven males of the same group was considered.

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:

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:

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.

Details on study schedule:

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.

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

Oestrous cyclicity (parental animals):

Oestrous cycle was monitored before treatment (pre-treatment period) to select for the study females with regular cyclicity.

Litter observations:

On Day 4 after birth, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four or five pups per sex. Blood samples were collected from two of the surplus pups, pooled, and stored for possible determination of T4 levels.
No pups were eliminated when litter size was dropped below the culling target (8 pups/litter).

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):

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. 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. All other males and females at all dosage levels survived to the scheduled necropsy without significant visible clinical signs.

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

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.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycle was monitored 14 days before treatment. The following females were excluded from the study - ID: 52, 57, 61, 73, 79, 81, 85, 93, 94, 103, 105, 106. Vaginal smears were evaluated on Day 13.

Reproductive performance:

no effects observed

Description (incidence and severity):

In summary none of used doses significantly influenced length of pregnancy, number of implants, survival of pups, anogenital distance, or other variables.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

In summary none of used doses significantly influenced length of pregnancy, number of implants, survival of pups, anogenital distance, or other variables.

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive 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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available