Reaction mass of (2R*,4R*,4aR*,9bS*)-2,4-dimethyl-4,4a,5,9b-tetrahydroindeno[1,2-d][1,3]dioxine and (2R*,4R*,4aS*,9bR*)-2,4-dimethyl-4,4a,5,9b-tetrahydroindeno[1,2-d][1,3]dioxine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Nov 2012 - 25 Oct 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 422 and GLP. During the study period the automatic light cycle of 12 h on/12 h off was not functional. Therefore, all animals were subjected to 24 h light.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 22 Mar 1996

Deviations:

yes

Remarks:

animals were subjected to 24 h light

GLP compliance:

yes (incl. QA statement)

Remarks:

Agence française de sécurité sanitaire des produits de santé, France

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dimethyl-2H,4H,4aH,5H,9bH-indeno[1,2-d][1,3]dioxine

EC Number:

944-528-8

Molecular formula:

C13H16O2

IUPAC Name:

2,4-dimethyl-2H,4H,4aH,5H,9bH-indeno[1,2-d][1,3]dioxine

Test animals

Species:

rat

Strain:

other: RccHan : WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 11 weeks
- Weight at study initiation: 311-368 g (males), 209-262 g (females)
- Housing: in groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops, during the pairing period, females were housed with sexually mature males (1:1), sterilized standard softwood was used as bedding with paper enrichment
- Diet: pelleted standard Harlan Teklad 2018C rodent maintenance diet (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: community tap-water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 0/24

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily using the test substance as supplied and weekly if stability of the test substance for 8 days was confirmed. Separate formulations were prepared for each concentration. Homogeneity of the test substance in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 2, 8 and 40 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Batch no.: 332190038, 382191749 and 492194511

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days) samples of about 1 g of each concentration were taken from the middle of each aliquot used on Day 7 of the treatment. On two further time points, samples were taken from the middle to confirm concentration. The samples were analyzed by GC/FID following an analytical procedure. The test substance was used as the analytical standard.
The test substance concentrations in the dose formulations ranged from 81.4% to 115.2% with reference to the nominal and were within the accepted range of ±20%. The homogeneous distribution of the test substance in the preparations was approved because single results found did not deviate more than 4.4% from the corresponding mean and met the specified acceptance criterion of ≤15%.
In addition, the test substance was found to be stable in application formulations when kept 8 days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.

Duration of treatment / exposure:

males: for at least 28 days, starting 2 weeks before mating and a maximum of 14 days during mating
females: for 2 weeks prior to mating, throughout gestation until the F1 generation reached Day 3 post partum (one day before necropsy)

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 40 and 200 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Main groups: initially 11
Additional: 6 at 0 and 40 mg/kg bw/day; 8 at 10 mg/kg bw/day

Because of the low percentage of mated females, additional animals were assigned to the control, low and mid dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats using dose levels of 0, 50, 150 and 500 mg/kg bw/day. The study was performed in the same laboratory and by the same study director in 2012. Four groups of 5 males and 5 females were treated by gavage with the test substance once daily over a 14-day period. At 500 mg/kg bw/day, two females were found dead on Day 4 and one male and one female were found dead on Day 5. The remaining males and females at this dose level were sacrificed on Day 5 of the treatment period for ethical reasons. At 50 and 150 mg/kg bw/day, all males and females survived until the scheduled necropsy. At 500 mg/kg bw/day, mean food consumption and body weight gain were statistically significantly reduced in the males and females up to necropsy. At 150 mg/kg bw/day, mean food consumption in males was slightly but not statistically significantly reduced during the treatment period. No test substance-related macroscopical findings were observed in the males and females at any dose level. Based on these observations, dose levels of 10, 40 and 200 mg/kg bw/day were considered to be suitable for the main study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: mortality/viability
Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed outside the home cage in all animals. In males, it was performed once prior to the first administration of the test substance and weekly thereafter. In females, it was performed once prior to the first administration of the test substance, weekly during the pre-pairing and pairing periods and on Days 0, 6, 13 and 20 of the gestation period. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION: Yes
- Food consumptions of males were recorded on pre-pairing period days 1-4, 4-8, 8-11 and 11-13 and after the pairing period weekly. Food consumptions of females were recorded on pre-pairing period days 1-4, 4-8, 8-11 and 11-13, on gestation days 0-7, 7-14 and 14-21 and on days 1-4 of the lactation. Food consumption was not recorded during the pairing period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 14 of the pre-pairing period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (18h)
- How many animals: 5 males and females from each group
- Parameters examined: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, total leukocyte count, differential leukocate count, platelet count, prothrombin time (thromboplastin time), activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 14 of the pre-pairing period
- Animals fasted: Yes (18 h)
- How many animals: 5 males and females from each group
- Parameters examined: glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl-transferase, bile acids, sodium, potassium, chloride, calcium, phosphorus, total protein, albumin, globulin, albumin/globulin ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at one time during the study (males shortly before the scheduled sacrifice and females on Day 3 post partum)
- Dose groups that were examined: 5 males and females from each group
- Battery of functions tested:
cage-side observations: faeces-balls, urine and posture as well as resistance to removal
hand-held observations: muscle tone, constitution, skin, pupil size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities
open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine
reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex)
measurements/counts: hind limb / fore limb grip strength, locomotor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Males were sacrificed after treatment for at least 28 days, when no longer needed for the assessment of reproductive effects. The additional males were terminated following mating of the respective females. Dams and pups were sacrificed on day 4 post partum. If birth did not occur on the expected date (Day 21 post coitum), the dam was sacrificed on Day 25 post coitum.

All parent animals and pups, except those excessively cannibalized and the additional parental males, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death.
For the parent animals, special attention was directed at the organs of the reproductive system. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

At the scheduled sacrifice, the following organs were weighted (*) and preserved in neutral phosphate buffered 4% formaldehyde solution: epididymides* (fixed in Bouin's solution), ovaries, prostate gland and seminal vesicles incl. coagulating glands, testes* (fixed in Bouin's solution), adrenal glands*, bone marrow (femur), brain - including section of medulla/pons*, cerebral and cerebellar cortex, cecum, colon, duodenum, heart including auricles*, ileum (with Peyer's patches), jejunum (with Peyer's patches), kidneys*, liver*, lungs (preserved by inflation with fixative and then immersion), lymph nodes – mesenteric and mandibular, rectum, sciatic nerve, spinal cord - cervical, midthoracic, lumbar, spleen*, stomach, thymus*, thyroid (incl. parathyroid gland, if possible), trachea, urinary bladder (preserved by inflation with fixative and then immersion), uterus (incl. oviducts, cervix and vagina), all gross lesions

HISTOPATHOLOGY: Yes
All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 µm and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.
Qualitative assessment of the male reproductive organs was performed. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

reduced food consumption in males and females at 200 mg/kg bw/day

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

increased ALAT level in males at 200 mg/kg bw/day

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased liver weights in males and increased liver and kidney weights in females at 200 mg/kg bw/day, decreased thymus weights in females at 200 mg/kg bw/day

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

increased incidence and severity of hyaline droplets in the renal tubular epithelium in males at 200 mg/kg bw/day, increased incidence of decreased lymphocytes in the thymus in females at 200 mg/kg bw/day

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
One female at 200 mg/kg bw/day was found dead on Day 3 of the lactation period. This female had ruffled fur, a hunched posture and decreased activity. At macroscopical examination, the thymus and mandibular lymph nodes in this female were reduced in size and there was red discoloration in the lungs. Microscopical examination revealed the cause of death to be attributed to bacterial contamination of the pregnant uterus/vagina with secondary systemic changes induced by thrombo-embolic bacterial dissemination and associated poor health status, unrelated to the test substance. All other males and females survived until the scheduled necropsy.

Findings at daily clinical observation:
On the first day of treatment reduced activity was observed in 5 females and 1 male at 200 mg/kg bw/day. Two females and one male had ptosis in both eyes. In the lactation period, one female had ruffled fur at 200 mg/kg bw/day. No clinical signs were observed at 40 mg/kg bw/day. At 10 mg/kg bw/day, one male had a wound on the tail apex in the pre-pairing and pairing period. No further clinical signs were observed.

Findings at detailed weekly clinical observations:
At all dose levels, there were incidences in males and females of delayed pupil reflex/absent iridic reflex, especially in the acclimatization period. This was considered to be a result of the lighting problem, to which the animals acclimatized. Incidences of salivation were observed in males and females at 200 mg/kg bw/day. Isolated incidences of swaying gait, decreased activity, increased activity, ruffled fur and Straub phenomenom were observed, which were considered to be incidental.

BODY WEIGHT AND WEIGHT GAIN
Males:
At 200 mg/kg bw/day, mean body weight gain was statistically significantly reduced from Day 2 until the end of the pre-pairing period. Over the whole pre-pairing period, the difference in mean body weight gain was 4% compared to 8% in the control group. Mean body weight was statistically significantly reduced from Day 7 of the pre-pairing period and remained so until the end of the study. The difference to the control group was 5% at 200 mg/kg bw/day at the end of the study. Mean body weight and body weight gain were not affected by treatment with the test substance at 10 and 40 mg/kg bw/day.
Females:
During the pre-pairing period mean body weight and body weight gain were statistically significantly reduced on Day 9 at 200 mg/kg bw/day. However, the difference to the control group was only slight (2%). During the gestation period, mean body weight and body weight gain were reduced without statistical significance (+44% compared to +53% in the control group). During the lactation period, although body weight gain was increased compared to the control group, mean body weight remained reduced. Mean body weight and body weight gain were not affected by treatment with the test substance at 10 and 40 mg/kg bw/day.

FOOD CONSUMPTION
Males:
At 200 mg/kg bw/day, mean food consumption was statistically significantly reduced during the pre-pairing period up to Days 8 - 11. Over the whole pre-pairing period, mean food consumption was -11.3% compared to the control group. At 10 and 40 mg/kg bw/day, mean food consumption was not affected by treatment with the test substance in the pre-pairing or after pairing periods.
Females:
At 200 mg/kg bw/day, mean food consumption was statistically significantly reduced up to Days 8 - 11 of the pre-pairing period (-14.5% over the whole of the pre-pairing period). It remained reduced during the gestation period and was statistically significantly reduced over the last 2 weeks. Over the whole of the gestation period, mean food consumption was -11.7% compared to the control group. During the lactation period, mean food consumption was similar to the control group. At 40 mg/kg bw/day, mean food consumption was similar to the control group during the pre-pairing period. During the gestation period, it was reduced without statistical significance over the last week (-9.4%). In the lactation period, it was increased compared to the control group. At 10 mg/kg bw/day, mean food consumption was not affected by treatment with the test substance.

HAEMATOLOGY
No test substance-related findings were observed at any dose level. The statistically significant changes observed were either not dose-dependent or were within the range of the historical control data.

CLINICAL CHEMISTRY
At 200 mg/kg bw/day, the alanine aminotransferase level was statistically significantly increased (+54.6%) in males. The remaining statistically significant changes observed were either not dose-dependent or were within the range of the historical control data. No test substance-related findings were observed at any dose level in females.

NEUROBEHAVIOUR
No test substance-related effects in males or females were observed at any dose level. Isolated findings included vocalization, increased number of faeces balls and decreased number of rearings. The body temperature and grip strength did not reveal any test substance-related effects. No effects on locomotor activity were observed at any dose level in males. In the females, locomotor activity in total was dose-dependently decreased. The decrease was not statistically significant and all values were within the range of the historical control data.

ORGAN WEIGHTS
At 200 mg/kg bw/day, in the males the absolute liver weight and body weight ratio were statistically significantly increased (12 and 20%, respectively). In addition, in the females the absolute weight, body weight ratios and brain weight ratios of the liver (22, 28 and 21%, respectively) and kidneys (22, 26 and 20%, respectively) were statistically significantly increased. However, no microscopical findings were found that correlated to changes in the liver and kidneys. In the females at 200 mg/kg bw/day, the absolute weight, body weight ratios and brain weight ratios of the thymus were statistically significantly decreased (45, 43 and 46%, respectively). This correlated with the reduced lymphocytes in the thymus, observed at microscopical examination. No test substance-related findings were observed in males and females at 10 and 40 mg/kg bw/day.

GROSS PATHOLOGY
No test substance-related findings were observed at any dose level in males. The thymus in one female was reduced in size at 200 mg/kg bw/day. This correlated with histopathological findings. No other test substance-related findings were observed at any dose level.

HISTOPATHOLOGY
In the males, there were no test substance-related findings in any examined reproductive organs. In particular, the qualitative examination of the stages of spermatogenesis in the testis did not reveal any test substance-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. The only potentially test substance-related finding was an increased incidence and severity of hyaline droplets in the renal tubular epithelium at 200 mg/kg bw/day. In the females, there were no test substance-related findings in the ovaries of non-pregnant females. In the pregnant females, there were no test substance-related microscopic findings in the reproductive organs, including following evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries. The only potentially test substance-related finding was an increased incidence of decreased lymphocytes in the thymus at 200 mg/kg bw/day. This was the histological correlate of the decreased thymus weight at 200 mg/kg bw/day when compared to control group. According to the authors, this was considered to be induced by the stress of pregnancy rather than a test substance-related finding.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

2

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced food consumption and increased liver weights (m, f)

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the NOAEL for systemic toxicity was set at 40 mg/kg bw/day due to effects on food consumption and increased liver weights at 200 mg/kg bw/day. No target organ was identified.