Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
OECD 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]benzoate
EC Number:
201-908-1
EC Name:
Methyl 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]benzoate
Cas Number:
89-43-0
Molecular formula:
C18H27NO3
IUPAC Name:
methyl 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]benzoate
Constituent 2
Reference substance name:
Reaction products of 7-hydroxy-3,7-dimethyloctanal and methyl 2-aminobenzoate
IUPAC Name:
Reaction products of 7-hydroxy-3,7-dimethyloctanal and methyl 2-aminobenzoate
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Aurantiol Pure (Product Code 2365901)
- Molecular formula (if other than submission substance): UVCB
- Molecular weight (if other than submission substance): 151 to 160 (range for identified components)
- Substance type: UVCB
- Physical state: Yellow viscous liquid
- Lot/batch No.:SC00010629
- Expiration date of the lot/batch: 26 November 2014
- Stability under test conditions: stable until the expiry date
- Storage condition of test material: Refrigerated and protected from light
- Volatility : Vapour pressure 0.4 Pa at 293K
Specific details on test material used for the study:
Appearance: Yellow viscous liquid
Batch: SC00020302
Purity/Composition: UVCB substance
Test item storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 22 February 2018 (expiry date)
Additional information
Test Facility test item number: 205387/D
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum-foil
Specific gravity/density 1.057,97 kg/m3 at 20°C
Chemical name (IUPAC), synonym or trade name Reaction products of 7-hydroxy-3,7-dimethyloctanal and methyl 2-aminobenzoate

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for ex trapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).
Sex:
male/female
Details on test animals or test system and environmental conditions:
On 17 May 2017, female Crl: WI(Han) rats were received and on 31 May 2017, male Crl:WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of administration, males were 10 weeks old and weighed between 261 and 287 g and females were 13 weeks old and weighed between 192 and 243 g.
A health inspection was performed before the initiation of administration.
The animals were allowed to acclimate to the Test Facility toxicology accommodation for 5 days prior to start of the pretest period (females) or 5 days before the commencement of dosing administration (males).
A total of 40 females was selected at randomization before initiation of the pretest phase. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study and their estrous cycle results were kept in the raw data but were not reported.
Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The oral route of administration via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test item intake was estimated based on the body weight and food consumption values.
The diet was provided in stainless steel containers, covered by a stainless steel grid to prevent spillage.
The same diets remained in the food hopper for a maximum of 1 day. During the daily weighing occasion, the remaining diet in the food hopper was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.
Details on mating procedure:
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females.
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that are littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.
Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item was mixed without the use of a vehicle, directly with the required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. Diets were prepared freshly for use at room temperature for a maximum of 1 day. Diets were kept in the freezer (≤-15°C) until use, if not used on the day of preparation for a maximum of 17 days.
All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility.
Residual samples were discarded after completion of the sample analysis.
Analyses were performed by by UPLC-UV using a validated analytical procedure (Test Facility Study No. 505073).
Duplicate sets of samples (approximately 5 g) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.
Duration of treatment / exposure:
The test item and control item were administered to the appropriate animals by inclusion in the diet ad libitum from Day 1 onwards for a minimum of 28 days. Males were exposed for 28 days, up to and including the day before scheduled necropsy. This included a minimum of two weeks prior to mating and during the mating period. Females that delivered were exposed for 50-63 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were exposed for 41-42 days.
The first day test diets were available to the animals was designated as Day 1.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test item intake was estimated based on the body weight and food consumption values.
Frequency of treatment:
Daily
Details on study schedule:
Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.
Live pups were weighed individually on PND 1, 4, 7 and 13.
Sex was externally determined for all pups on PND 1 and 4.
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
All male pups in each litter were examined for the number of areola/nipples on PND 13.
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 ppm
Dose / conc.:
3 000 ppm
Dose / conc.:
10 000 ppm
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
Prior to start of the pretest period (females) or treatment period (males), each animal was identified using earmark and tattoo. Prior to the pretest period, reserve females were numbered R1 through R8 at random by indelible marker.
Pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
A total of 40 females was selected at randomization before initiation of the pretest phase. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study and their estrous cycle results were kept in the raw data but were not reported.
Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.
On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes.

Positive control:
No

Examinations

Parental animals: Observations and examinations:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4).
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females.
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology.
Litter observations:
Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.
Live pups were weighed individually on PND 1, 4, 7 and 13.
Sex was externally determined for all pups on PND 1 and 4.
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
Postmortem examinations (parental animals):
Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. After collection all samples were transferred to the appropriate laboratory for analysis.
F0-animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.
Blood of F1-animals was collected on PND 4 and PND 13-15, if possible. This was performed in the necropsy room.

Blood samples were analyzed for the parameters:
White blood cells (WBC)
Neutrophil (absolute)
Lymphocyte (absolute)
Monocyte (absolute)
Eosinophil (absolute)
Basophil (absolute)
Red blood cells
Reticulocyte (absolute)


Blood samples were processed for plasma or serum (bile acids), which was analyzed for the parameters specified in the following
Alanine aminotransferase (ALAT)
Aspartate aminotransferase (ASAT)
Alkaline Phosphatase (ALP)
Total protein
Albumin
Total Bilirubin
Bile Acids
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate (Inorg. Phos)

The organs identified in the table below were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated
Brain
Cervixa
Epididymisb
Gland, adrenalb
Gland, coagulationb, c
Gland, parathyroidd
Gland, prostate
Gland, seminal vesicleb
Gland, thyroid
Heart
Kidneyb
Liver
Ovariesb
Spleen
Testesb
Thymus
Uterus


Postmortem examinations (offspring):
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND13-15), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
The pups selected for blood collection on PND 13-15 were anesthetized using isoflurane followed by exsanguination.
Pups that died before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. For details see also section 4.6.1.
All remaining pups were euthanized on PND 13-15. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
In addition, blood was collected from two pups per litter (see also section 4.6.1), and the thyroid from two pups per litter (one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
In addition, the thyroid was collected from two pups per litter (if possible, from one male and one female pup and from the same pups as selected for blood collection, see also section 4.6.1), and were preserved in 10% buffered formalin.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and \or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
For each group, the following calculations were performed. Group mean values were calculated from individual litter values.
Mating (%): Number of females mated x 100
Number of females paired

Precoital time: Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%): Number of pregnant females x 100
Number of females mated

Gestation index (%): Number of females with living pups on Day 1 x 100
Number of pregnant females

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%): Total number of offspring born x 100
Total number of uterine implantation sites


Live birth index (%): Number of live offspring on Day 1 after littering x 100
Total number of offspring born

Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check x 100
Number of live pups at First Litter Check

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check x 100
Number of live pups at First Litter Check

Viability index (%): Number of live offspring on Day 4 before culling x 100
Number live offspring on Day 1 after littering

Lactation index (%): Number of live offspring on Day 13 after littering x 100
Number live offspring on Day 4 (after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Incidental findings that were noted occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance.
Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post coitum and lactation phase.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were observed up to 10000 ppm.
Slightly lower body weights (not statistically significant) and body weight gain were noted for the males at 10000 ppm from Day 11 of treatment onwards. At the end of treatment, average body weight gain was 96% of the concurrent controls. Based on the slight change, lack of statistical significance and in the absence of correlating findings, this was considered to be non-adverse.
Body weights and body weight gain of males up to 3000 ppm and females up to 10000 ppm remained in the same range as controls over the treatment period.
The statistically significantly higher body weights that were noted for females at 1000 ppm on Day 8 of pre mating and during the post coitum phase were considered to be unrelated to treatment since the effect was small, no effects were seen for body weight gain and no trend was apparent regarding dose.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was slightly lower (without reaching statistical significance on most occasions) for females at 3000 and 10000 ppm during the post-coitum and for females at 10000 ppm also during the lactation phase. No dose response was observed, average values for relative food intake were 88% of controls for both dose levels during the post-coitum phase and 89% of controls during the lactation phase for females at 10000 ppm.
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for males up to 10000 ppm and females at 1000 ppm.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Reticulocyte levels were higher in females from 1000 ppm onwards, 18%, 30% and 36% increase relative to controls for the 1000 ppm, 3000 ppm and 10000 ppm groups, respectively. In females at 10000 ppm, the increase in reticulocyte levels was statistically significant, along with minimal decreases in red blood cell count, and haemoglobin and haematocrit levels and increases in red blood cell distribution width (all not statistically significant). All mean values remained well within the normal range for female rats of this strain and age. Reticulocyte levels were unaffected in males up to 10000 ppm.
Remaining haematological parameters were considered not to have been affected by treatment in males and females up to 10000 ppm.
Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes distinguished treated animals from control animals:
• Decreased total bilirubin in males at 10000 ppm (0.83x of controls).
• Slightly decreased chloride levels in males at 3000 and 10000 ppm (0.97x and 0.96x of controls, respectively).
• Slightly decreased total protein levels in females at 10000 ppm (0.94x of controls).
• Slightly increased creatinine levels in females at 10000 ppm (1.09x of controls). A similar trend was noted for males at 10000 ppm (1.07x of controls), but this difference was not statistically significant.
• Slight dose related increase in glucose levels in females (1.08x, 1.17x and 1.18x of controls for 1000, 3000 and 10000 ppm, respectively), but these differences were not statistically significant.
All values remained within the normal range for rats of this strain and age.
No toxicologically relevant changes were noted in clinical biochemistry parameters at dose levels at 1000 ppm in males and up to 3000 ppm in females.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
For males of all treatment groups, a slight decrease in T4 levels was observed; 0.89x, 0.90x and 0.83x of controls for 1000, 3000 and 10000 ppm treated males, respectively. All values remained within the historical control data .
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were not considered to be affected by treatment.
Grip strength of hind leg was slightly, but statistically significantly decreased for females at 10000 ppm. Values remained within the normal range for female rats of this strain and age.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with very high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no adverse test item-related microscopic observations.
There was a minor increase in incidence and severity of follicular cell hypertrophy in the thyroid gland of females at 10000 ppm (3/5 minimal, 1/5 slight) and of extramedullary hematopoiesis in the spleen of females at 10000 ppm (3/5 minimal, 1/5 slight), for which a relation to the treatment with the test item could not be excluded. As these findings were also recorded at minimal severity in 2/5 females of the control group and low severities can be seen as a background finding in females rats subjected to a combined 28-Day toxicity study, these were considered to be a non-adverse finding of no toxicological relevance.
For the remainder of the recorded microscopic findings there was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not considered to have been affected by treatment.
Most females had regular cycles of 4 to 5 days. Extended di-estrus occurred in female no. 54 (1000 ppm) with a regular cycle. Given the incidental nature, absence of a dose-related incidence, this finding did not indicate a relation with treatment.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was not considered to be affected by treatment. All females showed evidence of mating.
Precoital time was not considered to be affected by treatment. Most females showed evidence of mating within 4 days.
One female at 1000 ppm (no. 54) showed evidence of mating after 13 days only. Given the incidental nature and the absence of a dose-related incidence, this finding did not indicate a relation with treatment.
Number of implantation sites was not considered to be affected by treatment.
Fertility index was not considered to be affected by treatment.
A total of two females at 1000 ppm were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.
Gestation index and duration of gestation were not considered to be affected by treatment.
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
10 000 ppm
System:
other: Whole animals

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.
Note: Only days on which clinical signs were present between first and last litter check are presented in the report table.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment.
Three pups at 1000 ppm (one in litter 52, two in litter 55) and two pups at 3000 ppm (one in litter 63, one in litter 69) were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were not considered to be affected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
For the pup of female no. 69 (3000 ppm) who was found dead at first litter check, absence of milk in the stomach was noted at macroscopic examination. The nature and incidence of this and other macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.
Histopathological findings:
no effects observed
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
10 000 ppm
System:
other: Whole animals

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
10 000 ppm
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test an parental, reproduction and developmental no-observed-adverse-effect level (NOAEL) of at least 10000 ppm for Aurantiol Pure was established (corresponding to an overall mean test item intake of 710 and 1206 mg/kg bw/day for males and females, respectively).
Executive summary:

No reproduction toxicity was observed up to the highest dose level tested (10000 ppm).

No treatment-related toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

No developmental toxicity was observed up to the highest dose level tested (10000 ppm).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

For the exposure scenarios assessment and DNELs, the male NOAEL has been chosen.