Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The NOAEL for reproductive toxicity was considered to be 150 mg/kg/d upon oral gavage administration in rats, based on a OECD 421 study with a duration of 4 -6 weeks.

Link to relevant study records

Referenceopen allclose all

Endpoint:
reproductive toxicity, other
Remarks:
OECD 407 results
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 407
Deviations:
yes
Remarks:
see Principles of method if other than guideline; the study integrity was not adversely affected by the deviations.
Principles of method if other than guideline:
List of protocol deviations:
1. Temporary deviations from the maximum and minimum level of relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations.
2. The sciatic nerve of a control female (no. 23) was not available for histopathology. Evaluation: Sufficient tissues were available for evaluation of the results.
3. On days 3, 7, 9 and 23 the maximum time between the earliest and latest dosing exceeded 4 hours with a maximum of approximately 35 minutes. Evaluation: Deviation was of an incidental nature, and was not considered to have adversely affected the study results.
4. No arena observation was performed at the start of week 3. Evaluation: Based on the available arena observations and clinical observations, an adequate assessment of the study results was considered possible.

The study integrity was not adversely affected by the deviations.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Crl: (WI) BR (outbred, SPF-Quality)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: Charles River Deutschland, Sulzfeld, Germany.
Age at Start of treatment: Approximately 6 weeks.
Number of animals: 20 males, 20 females (females were nulliparous and non-pregnant)
Randomisation: Prior to commencement of treatment, by computer-generated random algorithm ac
cording to body weight, with all animals within +/- 20% of the sex mean.
Identification: Earmark and tattoo
Health inspection: A health inspection was performed prior to commencement of treatment to ensure t
hat the animals are in a good state of health.
Conditions: Animals were housed in a controlled environment, in which optimal conditions were co
nsidered to be approximately 15 air changes per hour, a temperature of 21.0 +/- 3.0 oC (actual range
19.7-22.8 oC), a relative humidity of 30-70% (actual range 26-93%) and 12 hours artificial fluorescent
light and 12 hours darkness per day.
Accommodation: Group housing of 5 animals per sex in Macrolon plastic cages (MIV type, height 18
cm, during overnight activity monitoring individual housing in MIII type; height 15 cm) with sterilised
sawdust as bedding material (Woody-Clean type 3/4, Tecnilab-BMI BV, Someren, The Netherlands)
and paper as cage-enrichment (Enviro-dri, BMI, Helmond, The Netherlands). No cage-enrichment
was provided during overnight activity monitoring. Certificates of analysis were examined and then
retained in the NOTOX archives. Acclimatisation period was at least 5 days before start of treatment
under laboratory conditions.
Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage,
Germany). Each batch is analysed for nutrients and contaminants are analysed on a regular basis.
Results are examined and archived.
Water: Free access to tap water. Certificates of analysis (performed quarterly) were examined and
archived.
Results of analysis for ingredients and/or contaminants of bedding, diet and water were assessed and
did not reveal any findings that were considered to have affected study integrity.
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
Accuracy, homogeneity and stability over 5 hours of formulations of test substance in propylene glycol were demonstrated by analysis
Details on exposure:
Oral gavage, using a stainless steel stomach tube. Formulations were placed on a magnetic stirrer d
uring dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations prepared for use on day 3 were analysed to check homogeneity (highest
and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over
5 hours was also determined (highest and lowest concentrations). The analytical method used
was based on the results of a separate project for the development and validation of the analytical
method (NOTOX project 433136).
Analysis of the accuracy of dose preparations revealed values within the range of 90-100 % of
nominal (with one value of the group 2 formulation being slightly lower, i.e. 82% of nominal), which
was considered to represent an acceptable level of accuracy for formulations of this type.
Duration of treatment / exposure:
See frequency of treatment
Frequency of treatment:
Once daily for at least 28 days, 7 days per week, approximately the same time each day with a
maximum of approximately 4.5 hours difference between the earliest and latest dose. Animals were
dosed up to the day prior to necropsy.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
male and female
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
male and female
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
male and female
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
male and female
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Details on study design:
Based on the results of a 5-day range finding study NOTOX Project 433226), the dose levels for the 2
8-day toxicity study were selected to be 0, 15, 150 and 450 mg/kg/day.
Dose volume: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the
latest body weight.
Parental animals: Observations and examinations:
Mortality/Viability: At least twice daily. Animals showing pain, distress or discomfort, which was
considered not transient in nature or was likely to become more severe, were sacrificed for humane
reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/2000/7). The
time of death was recorded as precisely as possible.

ior to start of treatment and on a weekly basis thereafter (except inadvertently at start of week 3), this
was also performed outside the home cage in a standard arena. The symptoms were gradred ac
cording to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe
Functional Observations: During week 4 of treatment, the following tests were performed on all an
imals (abbreviations mentioned in the respective tables indicated between brackets):
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip st
rength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a com
puterised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Body weights: On days 1, 8, 15, 22 and 28.
Food consumption: Weekly.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative
investigation introduced as no effect was suspected.
Oestrous cyclicity (parental animals):
Cervix, clitoral gland, ovaries, uterus and vagina were collected from all animals at necropsy and fixed in a neutral phophate buffered 4% formaldehyde solution
Sperm parameters (parental animals):
Prostate gland, seminal vesicles and testes were collected from all animals at necropsy and fixed in a neutral phophate buffered 4% formaldehyde solution
Litter observations:
n/a
Postmortem examinations (parental animals):
All animals surviving to the end of the observation period and all moribund animals were deeply anae
sthetised using iso-flurane and subsequently exsanguinated. All animals assigned to the study were
necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following t
issues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buff
ered 4% formaldehyde solution:
Identification marks: not processed Ovaries
Adrenal glands Pancreas
Aorta Peyer's patches [jejunum, ileum] if detectable
Brain [cerebellum, mid-brain, cortex] Pituitary gland
Caecum (Preputial gland)
Cervix Prostate gland
(Clitoral gland) Rectum
Colon (Salivary glands - mandibular, sublingual)
Duodenum Sciatic nerve
Epididymides (Seminal vesicles)
(Eyes with optic nerve [if detectable] and (Skeletal muscle)
Harderian gland) (Skin)
(Femal mammary gland area) Spinal cord - cervical, midthoracic, lumbar
(Femur including joint) Spleen
Heart Sternum with bone marrow
Ileum Stomach
Jejunum Testes
Kidneys Thymus
(Larynx) Thyroid including parathyroid [if detectable]
(Lacrimal gland, exorbital) (Tongue)
Liver Trachea
Lung, infused with formalin Urinary bladder
Lymph nodes - mandibular, mesenteric Uterus
(Nasopharynx) Vagina
Oesophagus All gross lesions
Tissues mentioned within brackets were not examined microscopically as there were no signs of toxicity or target organ involvement
Postmortem examinations (offspring):
n/a
Reproductive indices:
n/a
Offspring viability indices:
n/a
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All surviving females and one male at 450 mg/kg/day showed an abnormal gait during the last week
of treatment. One female sacrificed on day 3 showed clonic spasms prior to sacrifice. In addition, a
bdominal swelling was observed for one high dose male in week 4. Salivation was observed among
all animals at 150 and 450 mg/kg/day, and among one male at 15 mg/kg/day.
Laboured respiration and rates were shown be the female at 15 mg/kg/day sacrificed on day 6, and
were considered to have occurred due to a gavage accident. Other (incidental) findings that were
noted included alopecia on the head, and swelling and dark colouration of the right eye. These findin
gs are incidentally noted in rats of this age and strain, which are housed and treated under the co
nditions in this study. At the incidence observed, these were considered signs of no toxicological
significance. No clinical signs were noted in the control group.
Mortality:
mortality observed, treatment-related
Description (incidence):
Three females at 450 mg/kg/day (nos. 36, 38 and 40) were found dead or were sacrificed for humane
reasons between days 3 and 19.

One female at 15 mg/kg/day (no. 30) was sacrificed for humane reasons on day 6. Histopathology
revealed necrosis of the tracheal epithelia, which in combination with breathing difficulties seen prior
to sacrifice (laboured respiration and rates) was considered indicative of a gavage accident. Also, no
further mortality occurred in this dose group or the next higher dose group of 150 mg/kg/day.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in body weights and body weight gain were observed over the 4-
week study period.
Higher body weights and body weight gain levels were recorded for males at 15 mg/kg/day th
roughout treatment (achieving a level of statistical-significance in most instances). Taking into
account the nature of the effect (i.e. an increase) and since mean body weight (gain) of males at 1
50 and 450 mg/kg/day was similar to control levels, these changes were considered to be of no toxic
ological significance. Body weights and body weight gain of the other treated animals remained in the
same range as control animals during the treatment phase.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Relative food intake was slightly reduced for high dose females in weeks 1 and 2.
Food consumption before or after allowance for body weight was similar between other treated and
control animals.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes distinguished treated from control animals:
- Increased red blood cell distribution width (RDW) in ales and females at 450 mg/kg/day;
- Reduced red blood cell counts (RBC) in females at 150 and 450 mg/kg/day;
- Reduced haemoglobin level (HGB) in females at 150 and 450 mg/kg/day;
- Reduced haematocrit level (HCT) in females at 150 and 450 mg/kg/day;
- Reduced mean corpuscular haemoglobin concentration (MCHC) in females at 450 mg/kg/day;
- Increased reticulocyte counts in males and females* at 450 mg/kg/day;
- Reduced prothrombin time (PT) in females at 450 mg/kg/day
*no statistical significance.
Minor statistically significant differences included increased platelet counts and lower white blood
cell count in males and femals at 150 mg/kg/day respectively. These findings were considered to
have arisen by chance and in the absence of a treatment-related distribution considered to be of no
toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) differences were noted between control animals and treated
animals:
- Increased urea level in males and females* at 450 mg/kg/day;
- Increased creatinine levels in males at 450 mg/kg/day;
- Increased potassium level in males at 150 and 450 mg/kg/day, and females at 150 and 450* mg/kg/
day;
- Increased inorganic phosphate level in males and females* at 450 mg/kg/day;
- Increased cholesterol levl in females at 450 mg/kg/day*
*no statistical significance.

The lower alkaline phosphatase (ALP) level in males at 150 mg/kg/day was considered to be of no
toxicological significance, since no dose-related response was apparent and taking into account the
nature of the effect (i.e. a decrease).
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals, survi
ving to the end of the study. The variation in motor activity did not indicate a relation with treatment.
The high value for the total high sensor readings of one high dose male no. 17 was considered to ha
ve occurred by chance and occurred in the absence of any supportive clinical signs. As this did not
form part of a group response, no toxicological relevance was ascribed to this change.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- Bile duct proliferation of minimal or slight degree (2/5 males and 2/5 females at 150 mg/kg/day, 4/5
males and 4/5 females at 450 mg/kg/day);
- Diffuse midzonal/centrilobular hypertrophy of the liver at minimal or slight degree (4/5 males at 450
mg/kg/day);
- Increased severity of cortical hyaline droplets in the kidneys to slight or moderate degree (4/5 males
at 150 mg/kg/day and 5/5/ males at 450 mg/kg/day);
- Very slight increase in the severity of splenic hemopoiesis (primarily erythropoiesis) to a moderate
degree (1/5 males at 450 mg/kg/day).
All other microscopic findings were within the range of background pathology encountered in Wistar
rats of this age and strain and occurred at similar incidences and severity in both control and treated
rats.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
No effects observed in cervix, clitoral gland, ovaries, uterus and vagina
No effects observed in prostate gland, seminal vesicles and testes
Remarks on result:
not measured/tested
Remarks:
No effects observed in cervix, clitoral gland, ovaries, uterus and vagina or in prostate gland, seminal vesicles and testes
Remarks on result:
not measured/tested
Reproductive effects observed:
no
Conclusions:
In an OECD 407 study, there was no treatment related histopathological effects in cervix, clitoral gland, ovaries, uterus and vagina or in prostate gland, seminal vesicles and testes
Executive summary:

In an OECD 407 study, there was no effect seen in cervix, clitoral gland, ovaries, uterus and vagina or in prostate gland, seminal vesicles and testes upon histopathologic examination.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 15 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Please see any other information on materials and methods section
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Identification: 3,3,5,7,7-pentamethyl-1,2,4-trioxepane
Appearance: Clear Liquid
CAS Number: 215877-64-8
Sponsors Identification: Tx 311
Product Name: Trigonox 311
Supplier: Akzo Nobel Functional Chemicals b.v.
Product Number: 61164
EC Name: 3,3,5,7,7-pentamethyl-1,2,4-trioxepane
Chemical Name: 3,3,5,7,7-pentamethyl-1,2,4-trioxepane
Purity: 97.9% 3,3,5,7,7-pentamethyl-1,2,4-trioxepane
Batch Number: 1711423006
Label: TRIGONOX 311, 3,3,5,7,7-pentamethyl-1,2,4-trioxepane
Date Received: 20 December 2017
Storage Conditions: Room temperature in the dark
Expiry Date: 20 December 2019
No correction for purity was made.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for twenty days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 281 to 356g, and were approximately eleven weeks old. The females weighed 188 to 255g, and were approximately fourteen weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 6. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Propylene Glycol. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for at least twenty-one days. Formulations were therefore prepared fortnightly and stored at approximately 4 °C in the dark.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Propylene Glycol.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals
Details on mating procedure:
See details on study procedures, below.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken on two occasions and analyzed for concentration of 3,3,5,7,7-pentamethyl-1,2,4-trioxepane at Envigo Research Limited, Shardlow, UK, Analytical Services.

The test item concentration in the test samples was detemined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Preparation of calibration standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.2 mg/mL.
On each occasion, standard solutions derived from two stock standard solutions were used for calculation.
Calibration solutions were injected into the instrument, at the beginning and end of each sample analysis as a minimum, using the conditins detailed in the instrument parameters described below.
To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solutiopn of 1 mg/mL by serial dilution covering the concentration range 0.0523 mg/mL to 0.1569 mg/mL.

Preparation of test samples
The formulations received were extracted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent, this was then shaken to dissolve. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

Preparation of accuracy and precision samples
Samples of diltilled water were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis.
The concentration of Test Item in the final solution was quantified by gas chromatography using flame ionisation detection.

Instrument parameters
GC system: Agilent Technologies 6890, incorporating autosampler and workstation
Column: DB-5 (30 m x 0.53 mm id x 5 μm film)
Oven temperature programme: Oven: 100°C for 0 minutes with 10°C/minute to 250°C for 0 minutes
Injection temperature: 250°C
Flame ionisation detection temperature: 250°C
Injection volume: 1.0 μL
Retention time: -4 mins

Method Validation
The analytical procedure was successfully validated for Test item in Propylene glycol with respect to the specificity of chromatographic analysis, the linearity of detector response, method accuracy and precision. Reults are summarized below:
The specifity of the analytical method was demonstrated by the absence of a peak at the characteristic retention time for Test Item in the control sample chromatogram.
The calibration data for the calibration standards of the test item was found to have a linear correlation within the calibration range of 0.0523 mg/mL to 0.1569 mg/mL. The R2 fit of the calibration curve to the data was 0.9998, and was considered to be acceptable.
Method accuracy (recovery) and precision were confirmed. A mean recovery value of 101% (CV=0.629%, n=5) was obtained for 2.5 mg/mL and 99% (CV=0.485%, n=5) was obtained for 125 mg/mL.
The limit of quantification was determined as the lowest standard concentration used during the study.

Homogeneity and Stability of Dose Formulations
The homogeneity and stability if Test item in Propylene glycol formulation was assessed with respect to the level of concentration at nominal concentration of 2.5 mg/ML and 125 mg/mL.
Homogeneity was confirmed at the initial stability time point. The mean analysed concentration for the nine samples remained within 10% of the initial time zero value and the variation was less than 10%

Concentration of Dose Formulations
The mean concentrations were within applied ±10%, confirming the accurate formulation.

Conclusion
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for Test item in Propylene glycol formulations at nominal concentrations of 2.5 mg/mL and 125 mg/mL when stored refrigerated for 21 days.
The mean concentration of Test Item in test formulation analysed for the study were within ±10% of nominal concentrations, confirming the accurate formulation.
Duration of treatment / exposure:
6 weeks males and 9 weeks females (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Frequency of treatment:
Daily
Details on study schedule:
Chronological Sequence of Study
i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.
ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study. For the 14 days prior to pairing, pre-pairing vaginal smears were performed and assessed for females.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance, visible nipple counts (male offspring) and clinical signs were also recorded during this period.
vi. On Day 4 post partum, where possible, blood sampling was performed on two randomly allocated offspring from each litter in order to obtain serum samples.
vii. The male dose groups were killed and examined macroscopically on Day 44 or 45.
viii. On Day 13 post partum, where possible, blood sampling to produce serum samples for assessment of thyroid hormones was performed on two randomly selected offspring (one male and one female) per litter. Where possible, a further two randomly selected offspring (one male and one female) per litter were sampled to produce plasma samples. Thyroid/parathyroid samples were also retained from one male and one female from each litter where litter sizes allowed. All surviving offspring were killed and examined externally; where external observations were detected an internal necropsy was performed.
ix. All females were sacrificed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all females in the morning of the day of necropsy. Any female which did not produce a pregnancy was also sacrificed and examined macroscopically around the same time as littering females. In addition, blood samples to produce both serum and plasma were taken from all adult animals at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 males and females per dose group and control group
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen in collaboration with the Sponsor and were based on the results of previous toxicity work. Findings in the previous study included mortality at 450 mg/kg bw/day and microscopic liver changes at 450 and 150 mg/kg bw/day.
Parental animals: Observations and examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded for all animals at terminal kill.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7, 7-14.
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males through-out the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrous or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.
Sperm parameters (parental animals):
Testes weight and histopathology
Litter observations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.
Postmortem examinations (parental animals):
Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 14 post partum. Any females which failed to achieve pregnancy or produce a litter were killed around the same time as littering females.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Thyroid Hormone Analysis
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
Where possible serum samples were taken from two randomly allocated offspring from each litter on Day 4 post partum (if offspring were of the same sex, samples from the same litter were pooled). If eight or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
All serum samples were dispatched to the Test Site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) where the serum from adult males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator (I Komjarova). All plasma samples were retained at the Test Facility.

Organ Weights
The epididymides, testes, seminal vesicles (with coagulating gland) and prostate were removed from terminal kill adult males, dissected free from fat and weighed before fixation. Thyroid/parathyroid and liver were dissected free from fat for terminal kill animals from both sexes, and were weighed prior to fixation for the liver and following partial fixation for thyroid/parathyroid.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Epididymides (preserved in Modified Davidsons fluid)
Pituitary
Glans penis
Prostate
Gross lesions
Seminal Vesicles (with coagulating gland)
LABC (levator ani-bulbocavernous) muscle
Testes (preserved in Modified Davidsons fluid)
Liver
Thyroid/Parathyroid
Mammary gland
Uterus/Cervix (with oviducts)
Ovaries
Vagina
Where possible on Day 13 of age, for one male and one female offspring per litter, the thyroid/parathyroids were retained in 10% Buffered Formalin.
All tissues were dispatched to the histology processing Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU, UK) for processing (Principal Investigator: J Jones). The tissues from control and 150 mg/kg bw/day dose group animals and any animals which did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from all control and 150 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings observed was conducted by V Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS.
Postmortem examinations (offspring):
Necropsy
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation.
Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed; then an additional internal examination was performed.

Thyroid Hormone Analysis
Where possible, serum samples were taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. Where possible, plasma samples were also taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. If required the number/sex of offspring sampled was altered depending on the litter constituents.
Statistics:
Please refer to "Any other information on materials and methods including tables"
Reproductive indices:
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated / Nymber of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring) / Number of pregnant females) x 100
Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calcula ted for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).
i. Implantation Losses (%)
Group mean percentile post-implantation loss was calculated for each female/litter as follows:
Post–implantation loss (%) = ((Number of implanations on sites - Total number of offspring born) / Number of offspring born) x100
ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100
Viability Index 2 (%) = (Number of offspring alive on Day 13 / Number of offspring alive on Day 4) x 100
Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post partum.
iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4, 7 and 13 post partum, using the following formula:
(Nummber of male offspring / Total number of offpsring) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Noisy respiration and increased salivation were evident in the majority of animals of either sex treated with 150 and 50 mg/kg bw/day throughout the treatment period. These observations were also evident in animals of either sex treated with 15 mg/kg bw/day albeit to a lesser extent. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and reflect the distaste of the formulation or potential difficulty in dosing particular animals on occasions rather than true systemic toxicity.
One female treated with 150 mg/kg bw/day had a decreased respiratory rate, hunched posture and pilo-erection on Day 40. This was around the time of this female’s parturition, therefore, these observations were considered not to be related to treatment. Two males treated with 150 mg/kg bw/day had an open wound and subsequent scab formation between Days 31 and 43. Observations of this nature are commonly observed in group housed males and are considered to be incidental.
One control male had red/brown staining around the eyes on two occasions and one control female had red/brown staining around the ano-genital region on one day. These were considered to be incidental.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effect in body weight development was evident in treated males.
No effect in body weight development was evident in treated females during maturation, gestation or lactation.
Males treated with 15 mg/kg bw/day showed a statistically significant increase (p<0.05) in body weight gain during Week 4 and males from all treatment groups showed statistically significant increases (p<0.05) in body weight gain during Week 6. Females from all treatment groups showed a statistically significant increase (p<0.05) in body weight gain during the first week of treatment and females treated with 150 mg/kg bw/day also showed a statistically significant increase (p<0.05) in body weight gain between Days 4 and 7 of lactation. An increase in body weight gain is considered not to represent an adverse effect of treatment
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption for males throughout the pre-pairing and post-pairing phases of the study at 15, 50 or 150 mg/kg bw/day.
There was no effect of treatment on food consumption for females during the pre-pairing, gestation or lactation phases of the study at 15, 50 or 150 mg/kg bw/day.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no effect of treatment on food conversion efficiency for males throughout the pre-pairing and post-pairing phases of the study at 15, 50 or 150 mg/kg bw/day.
There was no effect on food conversion efficiency for females during pre-pairing at 15, 50 or 150 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on water consumption at 15, 50 or 150 mg/kg bw/day.
Daily visual assessment of water consumption did not reveal any significant intergroup differences
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Evaluation of Thyroxine (T4) in adult males at Day 13 of age did not identify any adverse effect of treatment or indication of endocrine disruption at 15, 50 or 150 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities detected.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Assessment of estrous cycles during the pre-pairing phase of the study and at termination did not indicate any obvious effect of treatment at 15, 50 or 150 mg/kg bw/day. All females showed regular cycling during the pre-pairing phase. There were also no intergroup differences in the stage of estrus on the day of necropsy
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle)
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating
Mating performance as assessed by the number of paired animals that mated was unaffected by treatment at dosages of 15, 50 or 150 mg/kg bw/day.

Fertility
There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy, at dosages of 15, 50 or 150 mg/kg bw/day.
One control female, two females treated with 50 mg/kg bw/day and two females treated with 150 mg/kg bw/day showed positive evidence of mating but were non-pregnant. With the exception of one of the male partners at 150 mg/kg bw/day which showed marked, unilateral tubular atrophy in the testes, no other changes were noted at histopathology in either the females or the males to account for these lack of pregnancies.

Gestation Length
The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 15, 50 or 150 mg/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
water consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs apparent for the offspring during the study were generally typical of the age observed and neither the distribution nor incidence of these findings indicated any effect of maternal treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
In litters from females treated with 150 mg/kg bw/day, live birth index and subsequent offspring survival to Day 13 of age was lower than control litters. Statistical significance (p<0.05) was evident for offspring viability on Day 4 and in overall viability. Litter size at 150 mg/kg bw/day, however, was higher than control litters at birth and during lactation and therefore the perceived differences compared to controls do not represent an adverse treatment related effect.

Females treated with 150 and 15 mg/kg bw/day showed a statistically significant reduction (p<0.05) in post-implantation loss. A reduction in this parameter actually represents better performance in the treated groups and is considered not to represent an adverse effect.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item indicated by offspring body weight or body weight gain
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Evaluation of Thyroxine (T4) in offspring at Day 13 of age did not identify any adverse effect of treatment or indication of endocrine disruption at 15, 50 or 150 mg/kg bw/day.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any obvious effect of maternal treatment at 15, 50 or 150 mg/kg bw/day.
Histopathological findings:
not examined
Description (incidence and severity):
There was no effect of treatment with the test item indicated by ano-genital distance on Day 1 post partum or visible nipple count in male offspring on Day 13 post partum at 15, 50 or 150 mg/kg bw/day.

Sex ratio for the offspring was similar to control in all treated groups and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology
Critical effects observed:
no
Reproductive effects observed:
no

Tabular summary report of effects on reproduction/development

Observations

Dose Level (mg/kg bw/day)

0 (Control)

15

50

150

Paired animals

n

12M 12F

12M 12F

12M 12F

12M 12F

Females showing evidence of copulation

n

12

12

12

12

Pregnant females

n

11

12

10

10

Conception Days 1-4

n

11

11

10

9

Conception Day 11

n

0

1

0

0

Conception Day 13

n

0

0

0

1

Gestation = 22 Days

n

2

0

1

0

Gestation = 22 ½ Days

n

5

8

4

6

Gestation = 23 Days

n

3

2

1

3

Gestation = 23 ½ Days

n

1

2

3

1

Gestation = 24 Days

n

0

0

1

0

Dams with live young born

n

11

12

10

10

Dams with live young at Day 13post partum

n

11

12

10

8

Implants/dam

x

13.1

11.3

13.2

13.8

Live offspring/dam at Day 1post partum

x

10.6

10.8

11.9

12.9

Live offspring/dam at Day 4 BCpost partum

x

10.5

10.4

11.9

12.3

Live offspring/dam at Day 4 ACpost partum

x

9.3

9.0

10.3

10.5

Live offspring/dam at Day 7post partum

x

9.3

9.0

10.3

10.3

Live offspring/dam at Day 13post partum

x

9.3

9.0

10.1

10.1

Sex ratio: % males at Day 1post partum

x

46.1

38.7

41.0

51.4

Sex ratio: % males at Day 4 BCpost partum

x

45.7

38.0

41.0

52.9

Sex ratio: % males at Day 4 ACpost partum

x

50.7

43.0

45.5

56.5

Sex ratio: % males at Day 7post partum

x

50.7

43.0

45.5

58.1

Sex ratio: % males at Day 13post partum

x

50.7

43.0

45.5

57.5

Litter weight (g) at Day 1post partum

x

59.91

62.46

69.62

73.93

Litter weight (g) at Day 4 BCpost partum

x

83.00

83.62

95.05

93.21

Litter weight (g) at Day 4 ACpost partum

x

73.62

72.84

82.94

79.76

Litter weight (g) at Day 7post partum

x

114.20

112.81

127.31

117.78

Litter weight (g) at Day 13post partum

x

221.19

213.48

238.83

231.30

Male offspring weight (g) at Day 1post partum

x

6.05

6.21

6.23

5.88

Male offspring weight (g) at Day 4 BCpost partum

x

8.57

8.59

8.51

7.76

Male offspring weight (g) at Day 4 ACpost partum

x

8.57

8.59

8.53

7.77

Male offspring weight (g) at Day 7post partum

x

13.22

13.22

13.03

11.64

Male offspring weight (g) at Day 13post partum

x

25.19

25.04

24.77

23.30

Female offspring weight (g) at Day 1post partum

x

5.67

5.92

5.87

5.58

Female offspring weight (g) at Day 4 BCpost partum

x

8.09

8.29

8.16

7.34

Female offspring weight (g) at Day 4 ACpost partum

x

8.05

8.29

8.18

7.28

Female offspring weight (g) at Day 7post partum

x

12.55

12.79

12.60

11.19

Female offspring weight (g) at Day 13post partum

x

24.26

24.38

23.91

22.64

LOSS OF OFFSPRING/DAM

 

 

 

 

 

Pre-natal (implantations minus live births)

 

 

 

 

 

0

n

2

9

2

5

1

n

4

0

4

2

2

n

0

2

3

1

3

n

3

1

1

1

6

n

1

0

0

0

8

n

1

0

0

0

n = Number

= Mean

Conclusions:
The oral administration of 3,3,5,7,7-pentamethyl-1,2,4-trioxepane to rats by gavage, at dose levels of 15, 50 and 150 mg/kg bw/day, did not result in any toxicologically significant effects up to 150 mg/kg bw/day. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study was therefore considered to be 150 mg/kg bw/day.
The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 150 mg/kg bw/day as the perceived differences to controls for live birth index and reduced offspring viability during lactation at 150 mg/kg bw/day are considered a consequence of higher group mean litter sizes at this dosage.
Executive summary:

Introduction

The study was performed to screen for potential adverse effects of 3,3,5,7,7-pentamethyl-1,2,4-trioxepane (CAS Number: 215877-64-8) on reproduction, including offspring development, to evaluate some endocrine disruptor relevant endpoints, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and nine weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 15, 50 and 150 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Propylene Glycol) over the same period.

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights ano-genital distance and visible nipple count (male offspring only).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Results

Adult Responses

Mortality

There were no unscheduled deaths on the study.

Clinical Observations

Incidences of noisy respiration and increased salivation were evident in animals of either sex treated with 150 mg/kg bw/day throughout the treatment period and to a lesser extent in animals of either sex treated with 50 and 15 mg/kg bw/day.

Body Weight

There was no effect of treatment on body weight or body weight gain of males throughout the study at 15, 50 and 150 mg/kg bw/day. There was no effect of treatment on body weight or body weight gain for females during pre-pairing, gestation or lactation phases at 15, 50 and 150 mg/kg bw/day.

Food Consumption and Food Conversion Efficiency

There was no effect of treatment on food consumption or food conversion efficiency for males throughout the study at 15, 50 and 150 mg/kg bw/day.

There was no effect of treatment on food consumption for females during pre-pairing, gestation or lactation phases at 15, 50 and 150 mg/kg bw/day. There was no effect on food conversion efficiency for females during pre-pairing at 15, 50 or 150 mg/kg bw/day.

Water Consumption

Visual inspection of water bottles throughout the study did not indicate any effect of treatment for either sex at 15, 50 or 150 mg/kg bw/day.

Reproductive Performance

Estrous Cycle

Assessment of estrous cycles during the pre-pairing phase of the study and at termination did not indicate any obvious effect of treatment at 15, 50 or 150 mg/kg bw/day.

Mating

There was no effect of treatment on mating at dosages of 15, 50 or 150 mg/kg bw/day.

Fertility

There was no effect of treatment on fertility at dosages of 15, 50 or 150 mg/kg bw/day.

Gestation Length

There was no effect of treatment on gestation length at dosages of 15, 50 or 150 mg/kg bw/day.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no effect of maternal treatment on the number of implantations, post-implantation loss or sex ratio at dosages of 15, 50 or 150 mg/kg bw/day.

In litters from females treated with 150 mg/kg bw/day, live birth index and subsequent offspring survival to Day 13 of age was lower than control litters. Litter size at 150 mg/kg bw/day, however, was higher than control litters.

Offspring Growth and Development

There was no effect of treatment with the test item indicated by offspring body weight or body weight gain, clinical signs, ano-genital distance on Day 1 post partum, or visible nipple count in male offspring on Day 13 post partum at 15, 50 or 150 mg/kg bw/day.

Pathology

Necropsy

Offspring

Macroscopic necropsy findings for offspring on the study did not indicate any effect of maternal treatment at 15, 50 or 150 mg/kg bw/day.

Adults

One female treated with 150 mg/kg bw/day had an enlarged liver at necropsy.

No such effects were detected in males treated with 150 mg/kg bw/day or in animals of either sex treated with 50 or 15 mg/kg bw/day.

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 15, 50 or 150 mg/kg bw/day.

Organ Weights

Animals of either sex from all treatment groups showed an increase in liver weight both absolute and relative to terminal body weight when compared to control animals.

Histopathology

There were no treatment-related microscopic abnormalities detected.

Conclusion

The oral administration of 3,3,5,7,7-pentamethyl-1,2,4-trioxepane to rats by gavage, at dose levels of 15, 50 and 150 mg/kg bw/day, did not result in any toxicologically significant effects up to 150 mg/kg bw/day. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study was therefore considered to be 150 mg/kg bw/day.

The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 150 mg/kg bw/day as the perceived differences to controls for live birth index and reduced offspring viability during lactation at 150 mg/kg bw/day are considered a consequence of higher group mean litter sizes at this dosage.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
Excellent (GLP)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

The NOAEL for developmental toxicity / teratogenicity was considered to be 150 mg/kg/d upon oral gavage administration in rats. This was based on a OECD 421 study with a duration of 4 -6 weeks.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 15 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Identification: 3,3,5,7,7-pentamethyl-1,2,4-trioxepane
Appearance: Clear Liquid
CAS Number: 215877-64-8
Sponsors Identification: Tx 311
Product Name: Trigonox 311
Supplier: Akzo Nobel Functional Chemicals b.v.
Product Number: 61164
EC Name: 3,3,5,7,7-pentamethyl-1,2,4-trioxepane
Chemical Name: 3,3,5,7,7-pentamethyl-1,2,4-trioxepane
Purity: 97.9% 3,3,5,7,7-pentamethyl-1,2,4-trioxepane
Batch Number: 1711423006
Label: TRIGONOX 311, 3,3,5,7,7-pentamethyl-1,2,4-trioxepane
Date Received: 20 December 2017
Storage Conditions: Room temperature in the dark
Expiry Date: 20 December 2019
No correction for purity was made.
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK.
On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for twenty days during which time their health status was assessed.
Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment.
A total of ninety six animals (forty eight males and forty eight females) were accepted into the study.
At the start of treatment the males weighed 281 to 356g, and were approximately eleven weeks old.
The females weighed 188 to 255g, and were approximately fourteen weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis.
Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water.
A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited Oxon, UK.) was used.
Certificates of analysis of the batches of diet used are given in Annex 6. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Propylene Glycol. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for at least twenty-one days. Formulations were therefore prepared fortnightly and stored at approximately 4 °C in the dark.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Propylene Glycol.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken on two occasions and analyzed for concentration of 3,3,5,7,7-pentamethyl-1,2,4-trioxepane at Envigo Research Limited, Shardlow, UK, Analytical Services.

The test item concentration in the test samples was detemined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Preparation of calibration standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.2 mg/mL.
On each occasion, standard solutions derived from two stock standard solutions were used for calculation.
Calibration solutions were injected into the instrument, at the beginning and end of each sample analysis as a minimum, using the conditins detailed in the instrument parameters described below.
To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solutiopn of 1 mg/mL by serial dilution covering the concentration range 0.0523 mg/mL to 0.1569 mg/mL.

Preparation of test samples
The formulations received were extracted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent, this was then shaken to dissolve. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

Preparation of accuracy and precision samples
Samples of diltilled water were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis.
The concentration of Test Item in the final solution was quantified by gas chromatography using flame ionisation detection.

Instrument parameters
GC system: Agilent Technologies 6890, incorporating autosampler and workstation
Column: DB-5 (30 m x 0.53 mm id x 5 μm film)
Oven temperature programme: Oven: 100°C for 0 minutes with 10°C/minute to 250°C for 0 minutes
Injection temperature: 250°C
Flame ionisation detection temperature: 250°C
Injection volume: 1.0 μL
Retention time: -4 mins

Method Validation
The analytical procedure was successfully validated for Test item in Propylene glycol with respect to the specificity of chromatographic analysis, the linearity of detector response, method accuracy and precision. Reults are summarized below:
The specifity of the analytical method was demonstrated by the absence of a peak at the characteristic retention time for Test Item in the control sample chromatogram.
The calibration data for the calibration standards of the test item was found to have a linear correlation within the calibration range of 0.0523 mg/mL to 0.1569 mg/mL. The R2 fit of the calibration curve to the data was 0.9998, and was considered to be acceptable.
Method accuracy (recovery) and precision were confirmed. A mean recovery value of 101% (CV=0.629%, n=5) was obtained for 2.5 mg/mL and 99% (CV=0.485%, n=5) was obtained for 125 mg/mL.
The limit of quantification was determined as the lowest standard concentration used during the study.

Homogeneity and Stability of Dose Formulations
The homogeneity and stability if Test item in Propylene glycol formulation was assessed with respect to the level of concentration at nominal concentration of 2.5 mg/ML and 125 mg/mL.
Homogeneity was confirmed at the initial stability time point. The mean analysed concentration for the nine samples remained within 10% of the initial time zero value and the variation was less than 10%

Concentration of Dose Formulations
The mean concentrations were within applied ±10%, confirming the accurate formulation.

Conclusion
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for Test item in Propylene glycol formulations at nominal concentrations of 2.5 mg/mL and 125 mg/mL when stored refrigerated for 21 days.
The mean concentration of Test Item in test formulation analysed for the study were within ±10% of nominal concentrations, confirming the accurate formulation.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrous or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Duration of treatment / exposure:
approximately six weeks (males) and up to nine weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Frequency of treatment:
Daily
Duration of test:
approximately six weeks (males) and up to nine weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 males/ 12 females per dose group and control group
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen in collaboration with the Sponsor and were based on the results of previous toxicity work. Findings in the previous study included mortality at 450 mg/kg bw/day and microscopic liver changes at 450 and 150 mg/kg bw/day.
Maternal examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded for all animals at terminal kill.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7, 7-14.
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males through-out the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Estrous Cycle Assessment
Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 14 post partum. Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Any females which failed to achieve pregnancy or produce a litter were killed around the same time as littering females.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed; then an additional internal examination was performed.

Thyroid Hormone Analysis
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
Where possible serum samples were taken from two randomly allocated offspring from each litter on Day 4 post partum (if offspring were of the same sex, samples from the same litter were pooled). If eight or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
Where possible, serum samples were taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. Where possible, plasma samples were also taken from two randomly allocated offspring per litter (one male and one female) on Day 13 post partum. If required the number/sex of offspring sampled was altered depending on the litter constituents.

Serum and plasma samples were taken from all adult males and females at termination.
All serum samples were dispatched to the Test Site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) where the serum from adult males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator (I Komjarova). All plasma samples were retained at the Test Facility.

Organ Weights
The epididymides, testes, seminal vesicles (with coagulating gland) and prostate were removed from terminal kill adult males, dissected free from fat and weighed before fixation. Thyroid/parathyroid and liver were dissected free from fat for terminal kill animals from both sexes, and were weighed prior to fixation for the liver and following partial fixation for thyroid/parathyroid.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Epididymides (preserved in Modified Davidsons fluid)
Pituitary
Glans penis
Prostate
Gross lesions
Seminal Vesicles (with coagulating gland)
LABC (levator ani-bulbocavernous) muscle
Testes (preserved in Modified Davidsons fluid)
Liver
Thyroid/Parathyroid
Mammary gland
Uterus/Cervix (with oviducts)
Ovaries
Vagina
Where possible on Day 13 of age, for one male and one female offspring per litter, the thyroid/parathyroids were retained in 10% Buffered Formalin.
All tissues were dispatched to the histology processing Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU, UK) for processing (Principal Investigator: J Jones). The tissues from control and 150 mg/kg bw/day dose group animals and any animals which did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from all control and 150 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings observed was conducted by V Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS.
Ovaries and uterine content:

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition
Fetal examinations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.
Statistics:
Please refer to "Any other information on materials and methods including tables"
Indices:
Please refer to "Any other information on materials and methods including tables"
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Noisy respiration and increased salivation were evident in the majority of animals of either sex treated with 150 and 50 mg/kg bw/day throughout the treatment period. These observations were also evident in animals of either sex treated with 15 mg/kg bw/day albeit to a lesser extent. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and reflect the distaste of the formulation or potential difficulty in dosing particular animals on occasions rather than true systemic toxicity.
One female treated with 150 mg/kg bw/day had a decreased respiratory rate, hunched posture and pilo-erection on Day 40. This was around the time of this female’s parturition, therefore, these observations were considered not to be related to treatment. Two males treated with 150 mg/kg bw/day had an open wound and subsequent scab formation between Days 31 and 43. Observations of this nature are commonly observed in group housed males and are considered to be incidental.
One control male had red/brown staining around the eyes on two occasions and one control female had red/brown staining around the ano-genital region on one day. These were considered to be incidental.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effect in body weight development was evident in treated males.
No effect in body weight development was evident in treated females during maturation, gestation or lactation.
Males treated with 15 mg/kg bw/day showed a statistically significant increase (p<0.05) in body weight gain during Week 4 and males from all treatment groups showed statistically significant increases (p<0.05) in body weight gain during Week 6. Females from all treatment groups showed a statistically significant increase (p<0.05) in body weight gain during the first week of treatment and females treated with 150 mg/kg bw/day also showed a statistically significant increase (p<0.05) in body weight gain between Days 4 and 7 of lactation. An increase in body weight gain is considered not to represent an adverse effect of treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption for males throughout the pre-pairing and post-pairing phases of the study at 15, 50 or 150 mg/kg bw/day.
There was no effect of treatment on food consumption for females during the pre-pairing, gestation or lactation phases of the study at 15, 50 or 150 mg/kg bw/day.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no effect of treatment on food conversion efficiency for males throughout the pre-pairing and post-pairing phases of the study at 15, 50 or 150 mg/kg bw/day.
There was no effect on food conversion efficiency for females during pre-pairing at 15, 50 or 150 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on water consumption at 15, 50 or 150 mg/kg bw/day.
Daily visual assessment of water consumption did not reveal any significant intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any adverse effect of treatment or indication of endocrine disruption at 15, 50 or 150 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex from all treatment groups showed a statistically significant increase (p<0.05-0.01) in liver weight both absolute and relative to terminal body weight when compared to control animals. A number of individual values were also outside of the normal expected ranges.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
One female treated with 150 mg/kg bw/day had an enlarged liver at necropsy.
No such effects were detected in males treated with 150 mg/kg bw/day or in animals of either sex treated with 50 or 15 mg/kg bw/day.
The following macroscopic abnormalities were detected, however, they were either present in the control group, did not follow a true dose related response or were not associated with any treatment-related findings in the treated groups and therefore were considered to be incidental. A mass (approximately 2 mm x 2 mm) on the right epididymis in one control male, pale areas on the liver in one control female and one female treated with 15 mg/kg bw/day, malformed kidney in one female treated with 15 mg/kg bw/day, increased renal pelvic space in the right kidney in one male treated with 50 mg/kg bw/day and in one male treated with 150 mg/kg bw/day and small and flaccid left testis and small left epididymis in one male treated with 150 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities detected.
There were no test item-related microscopic findings following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of follicles and corpora lutea in the ovaries.
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
Not applicable, as rats do not abort.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
There was no effect of maternal treatment on the number of implantations or post-implantation loss at dosages of 15, 50 or 150 mg/kg bw/day.

Females treated with 150 and 15 mg/kg bw/day showed a statistically significant reduction (p<0.05) in post-implantation loss. A reduction in this parameter actually represents better performance in the treated groups and is considered not to represent an adverse effect.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
Two females treated with 150 mg/kg bw/day exhibited a total litter loss.
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
live birth index was lower than control litters
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 15, 50 or 150 mg/kg bw/day.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy, at dosages of 15, 50 or 150 mg/kg bw/day.
One control female, two females treated with 50 mg/kg bw/day and two females treated with 150 mg/kg bw/day showed positive evidence of mating but were non-pregnant. With the exception of one of the male partners at 150 mg/kg bw/day which showed marked, unilateral tubular atrophy in the testes, no other changes were noted at histopathology in either the females or the males to account for these lack of pregnancies.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Mating performance as assessed by the number of paired animals that mated was unaffected by treatment at dosages of 15, 50 or 150 mg/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
clinical biochemistry
clinical signs
dead fetuses
effects on pregnancy duration
food consumption and compound intake
food efficiency
gross pathology
histopathology: non-neoplastic
maternal abnormalities
mortality
organ weights and organ / body weight ratios
pre and post implantation loss
water consumption and compound intake
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item indicated by offspring body weight or body weight gain, ano-genital distance on Day 1 post partum or visible nipple count in male offspring on Day 13 post partum at 15, 50 or 150 mg/kg bw/day.
Clinical signs apparent for the offspring during the study were generally typical of the age observed and neither the distribution nor incidence of these findings indicated any effect of maternal treatment.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of maternal treatment on the number of implantations or post-implantation loss at dosages of 15, 50 or 150 mg/kg bw/day.
Females treated with 150 and 15 mg/kg bw/day showed a statistically significant reduction (p<0.05) in post-implantation loss. A reduction in this parameter actually represents better performance in the treated groups and is considered not to represent an adverse effect.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio for the offspring was similar to control in all treated groups and did not indicate any selective effect of maternal treatment on survival for either sex at any of the dosages investigated.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Litter size at 150 mg/kg bw/day, however, was higher than control litters at birth and during lactation and therefore the perceived differences compared to controls do not represent an adverse treatment related effect.
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
In litters from females treated with 150 mg/kg bw/day, live birth index and subsequent offspring survival to Day 13 of age was lower than control litters. Statistical significance (p<0.05) was evident for offspring viability on Day 4 and in overall viability.
External malformations:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any obvious effect of maternal treatment at 15, 50 or 150 mg/kg bw/day.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
Developmental effects observed:
no

Tabular summary report of effects on reproduction/development

Observations

Dose Level (mg/kg bw/day)

0 (Control)

15

50

150

Paired animals

n

12M 12F

12M 12F

12M 12F

12M 12F

Females showing evidence of copulation

n

12

12

12

12

Pregnant females

n

11

12

10

10

Conception Days 1-4

n

11

11

10

9

Conception Day 11

n

0

1

0

0

Conception Day 13

n

0

0

0

1

Gestation = 22 Days

n

2

0

1

0

Gestation = 22 ½ Days

n

5

8

4

6

Gestation = 23 Days

n

3

2

1

3

Gestation = 23 ½ Days

n

1

2

3

1

Gestation = 24 Days

n

0

0

1

0

Dams with live young born

n

11

12

10

10

Dams with live young at Day 13post partum

n

11

12

10

8

Implants/dam

x

13.1

11.3

13.2

13.8

Live offspring/dam at Day 1post partum

x

10.6

10.8

11.9

12.9

Live offspring/dam at Day 4 BCpost partum

x

10.5

10.4

11.9

12.3

Live offspring/dam at Day 4 ACpost partum

x

9.3

9.0

10.3

10.5

Live offspring/dam at Day 7post partum

x

9.3

9.0

10.3

10.3

Live offspring/dam at Day 13post partum

x

9.3

9.0

10.1

10.1

Sex ratio: % males at Day 1post partum

x

46.1

38.7

41.0

51.4

Sex ratio: % males at Day 4 BCpost partum

x

45.7

38.0

41.0

52.9

Sex ratio: % males at Day 4 ACpost partum

x

50.7

43.0

45.5

56.5

Sex ratio: % males at Day 7post partum

x

50.7

43.0

45.5

58.1

Sex ratio: % males at Day 13post partum

x

50.7

43.0

45.5

57.5

Litter weight (g) at Day 1post partum

x

59.91

62.46

69.62

73.93

Litter weight (g) at Day 4 BCpost partum

x

83.00

83.62

95.05

93.21

Litter weight (g) at Day 4 ACpost partum

x

73.62

72.84

82.94

79.76

Litter weight (g) at Day 7post partum

x

114.20

112.81

127.31

117.78

Litter weight (g) at Day 13post partum

x

221.19

213.48

238.83

231.30

Male offspring weight (g) at Day 1post partum

x

6.05

6.21

6.23

5.88

Male offspring weight (g) at Day 4 BCpost partum

x

8.57

8.59

8.51

7.76

Male offspring weight (g) at Day 4 ACpost partum

x

8.57

8.59

8.53

7.77

Male offspring weight (g) at Day 7post partum

x

13.22

13.22

13.03

11.64

Male offspring weight (g) at Day 13post partum

x

25.19

25.04

24.77

23.30

Female offspring weight (g) at Day 1post partum

x

5.67

5.92

5.87

5.58

Female offspring weight (g) at Day 4 BCpost partum

x

8.09

8.29

8.16

7.34

Female offspring weight (g) at Day 4 ACpost partum

x

8.05

8.29

8.18

7.28

Female offspring weight (g) at Day 7post partum

x

12.55

12.79

12.60

11.19

Female offspring weight (g) at Day 13post partum

x

24.26

24.38

23.91

22.64

LOSS OF OFFSPRING/DAM

 

 

 

 

 

Pre-natal (implantations minus live births)

 

 

 

 

 

0

n

2

9

2

5

1

n

4

0

4

2

2

n

0

2

3

1

3

n

3

1

1

1

6

n

1

0

0

0

8

n

1

0

0

0

n= Number

= Mean

Conclusions:
The oral administration of 3,3,5,7,7-pentamethyl-1,2,4-trioxepane to rats by gavage, at dose levels of 15, 50 and 150 mg/kg bw/day, did not result in any toxicologically significant effects up to 150 mg/kg bw/day. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study was therefore considered to be 150 mg/kg bw/day.
The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive / developmental toxicity was considered to be 150 mg/kg bw/day as the perceived differences to controls for live birth index and reduced offspring viability during lactation at 150 mg/kg bw/day are considered a consequence of higher group mean litter sizes at this dosage.
Executive summary:

Introduction

The study was performed to screen for potential adverse effects of 3,3,5,7,7-pentamethyl-1,2,4-trioxepane (CAS Number: 215877-64-8) on reproduction, including offspring development, to evaluate some endocrine disruptor relevant endpoints, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 29 July 2016).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and nine weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 15, 50 and 150 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Propylene Glycol) over the same period.

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights ano-genital distance and visible nipple count (male offspring only).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Results

Adult Responses

Mortality

There were no unscheduled deaths on the study.

Clinical Observations

Incidences of noisy respiration and increased salivation were evident in animals of either sex treated with 150 mg/kg bw/day throughout the treatment period and to a lesser extent in animals of either sex treated with 50 and 15 mg/kg bw/day.

Body Weight

There was no effect of treatment on body weight or body weight gain of males throughout the study at 15, 50 and 150 mg/kg bw/day. There was no effect of treatment on body weight or body weight gain for females during pre-pairing, gestation or lactation phases at 15, 50 and 150 mg/kg bw/day.

Food Consumption and Food Conversion Efficiency

There was no effect of treatment on food consumption or food conversion efficiency for males throughout the study at 15, 50 and 150 mg/kg bw/day.

There was no effect of treatment on food consumption for females during pre-pairing, gestation or lactation phases at 15, 50 and 150 mg/kg bw/day. There was no effect on food conversion efficiency for females during pre-pairing at 15, 50 or 150 mg/kg bw/day.

Water Consumption

Visual inspection of water bottles throughout the study did not indicate any effect of treatment for either sex at 15, 50 or 150 mg/kg bw/day.

Reproductive Performance

Estrous Cycle

Assessment of estrous cycles during the pre-pairing phase of the study and at termination did not indicate any obvious effect of treatment at 15, 50 or 150 mg/kg bw/day.

Mating

There was no effect of treatment on mating at dosages of 15, 50 or 150 mg/kg bw/day.

Fertility

There was no effect of treatment on fertility at dosages of 15, 50 or 150 mg/kg bw/day.

Gestation Length

There was no effect of treatment on gestation length at dosages of 15, 50 or 150 mg/kg bw/day.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no effect of maternal treatment on the number of implantations, post-implantation loss or sex ratio at dosages of 15, 50 or 150 mg/kg bw/day.

In litters from females treated with 150 mg/kg bw/day, live birth index and subsequent offspring survival to Day 13 of age was lower than control litters. Litter size at 150 mg/kg bw/day, however, was higher than control litters.

Offspring Growth and Development

There was no effect of treatment with the test item indicated by offspring body weight or body weight gain, clinical signs, ano-genital distance on Day 1 post partum, or visible nipple count in male offspring on Day 13 post partum at 15, 50 or 150 mg/kg bw/day.

Pathology

Necropsy

Offspring

Macroscopic necropsy findings for offspring on the study did not indicate any effect of maternal treatment at 15, 50 or 150 mg/kg bw/day.

Adults

One female treated with 150 mg/kg bw/day had an enlarged liver at necropsy.

No such effects were detected in males treated with 150 mg/kg bw/day or in animals of either sex treated with 50 or 15 mg/kg bw/day.

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 15, 50 or 150 mg/kg bw/day.

Organ Weights

Animals of either sex from all treatment groups showed an increase in liver weight both absolute and relative to terminal body weight when compared to control animals.

Histopathology

There were no treatment-related microscopic abnormalities detected.

Conclusion

The oral administration of 3,3,5,7,7-pentamethyl-1,2,4-trioxepane to rats by gavage, at dose levels of 15, 50 and 150 mg/kg bw/day, did not result in any toxicologically significant effects up to 150 mg/kg bw/day. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study was therefore considered to be 150 mg/kg bw/day.

The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was considered to be 150 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
Excellent (GLP)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Description of key information

WoE: In an OECD 407 (oral repeated dose) study, there were no histopathological effects found in female (cervix, clitoral gland, ovaries, uterus and vagina) or in male (prostate gland, seminal vesicles and testes) reproductive organs.

Justification for classification or non-classification

Data are conclusive, but not sufficient for classification.

Additional information