6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-propionaldehyde

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 February - 17 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 February - 17 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks (males), 13-14 weeks (females)
- Weight at study initiation: 296 - 350 g (males), 195 and 249 g (females)
- Fasting period before study: No (except for males which were fasted overnight with a maximum of 24 hours before sacrifice; females were not fasted)
- Housing: 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). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water. The cages contained appropriate bedding.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum)
- Water: Municipal tap water, ad libitum
- Acclimation period: 6 days (females), 7 days (males)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed. It is considered that there were no known contaminants in the feed and in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 45-56
- Air changes (per hr): 10 or more air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 March 2020 To: 01 May 2020

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this route is expected to present the toxic effects best because full oral absorption is likely and route to route extrapolation is possible.

Vehicle:

unchanged (no vehicle)

Remarks:

Control animals received water.

Details on oral exposure:

The test item was administered as received. An adequate amount of the test item was dispensed into daily aliquots, which were stored the same as for the bulk test item until use. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the test item (0.9584 (D25/25)). No correction was made for the purity/composition of the test item.
The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. Dose volumes ≤ 50 µL were administered using a digital syringe and dose volumes > 50 µL were given with a plastic feeding tube connected to an appropriately graded syringe.
A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Dose volumes were 0.0313, 0.0626, 0.125 mL/kg bw for the low, mid and high dose groups, respectively. The control animals received 0.125 mL water/ kg bw.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The substance was dosed as such. Samples for dose formulation analysis were not collected.

Duration of treatment / exposure:

Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13 days of lactation (for 50-57 days). Females that failed to deliver pups were treated for 41-43 days.

Frequency of treatment:

Once daily, 7 days per week

Dose / conc.:

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

Remarks:

Low dose group

Dose / conc.:

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

Remarks:

Mid dose group

Dose / conc.:

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

Remarks:

High dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of a 14-day Dose Range Finder with oral administration of Pino Acetaldehyde in rats (Test Facility Reference No. 20137515) in which mortality was observed at 450 mg/kg bw/day but no severe effects were recorded at 90 mg/kg bw/day, and in an attempt to produce graded responses to the test item.
In the Dose Range Finder study significant treatment related changes were recorded for sperm parameters at 450 mg/kg bw/day (reduced mean motile sperm, mean progressive motility, mean number of cells with normal morphology and mean number of cells with coiled tail, and increased mean number of cells with detached head and mean number of cells with abnormal neck. Epididymal sperm count was not considered affected by treatment. These findings were not recorded at 90 mg/kg bw/day.

Positive control:

No.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for general health/mortality and moribundity
- The circumstances of any death were recorded in detail.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
- During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals (no peak effect of occurrence of clinical signs was observed in the dose range finder)
- The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.
- Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
The terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION: Yes
- Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Females were not fasted overnight.
- How many animals: 5/sex/group
- Parameters as specified in the OECD guideline were included (coagulation parameters (prothrombin time and activated partial thromboplastin time) were included.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Females were not fasted overnight.
- How many animals: 5/sex/group
- Parameters as specified in the OECD guideline were included
- Thyroid hormone level (total T4) was measured for males

FUNCTIONAL TESTS:
Functional tests were performed on 5 males/ group during Week 4 of treatment and 5 females/group during the last week of lactation (i.e. PND 6-13), These tests were performed after dosing, after completion of clinical observations (including arena observation, if applicable).
The following tests were performed:
- Hearing ability;
- Pupillary reflex;
- Static righting reflex;
- Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal;
- Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

ESTROUS CYCLE DETERMINATION
- 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 estrous.

Sacrifice and pathology:

Necropsy was conducted for animals that died on study, and specified tissues were saved.

Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

Organs (brain, cervix, epididymis, glands (adrenal, coagulation, parathyroid, prostate, seminal vesicle and thyroid), heart, kidney, liver, ovaries, spleen, testes, thymus and uterus) were weighed at necropsy for all animals at scheduled euthanasia. Organ weights were not recorded for animals found dead. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

Histopathological examination of all major tissues as defined in the OECD guidelines was performed for 5 animals/sex in the control and high dose groups and for animals found dead. In addition the liver of 5 animals (both sexes) in the low and the mid dose groups were histopathologically examined, based on possible treatment-related changes in this tissue.
For males that failed to sire (except for males which were selected) and females that failed to deliver pups the reproductive tissues (cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina) were histopathologically examined.
All gross lesions/masses found were histopathologically examined.
For the testes of 5 males of the control and the high dose group and for all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

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% 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 3 observations.
The following pairwise comparisons were made:
Low dose group vs. control group, mid dose group vs. control group, high dose group vs. control group.

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).

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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- See Table 1.1 (Page 44-45) of the attached full study report -
No adverse treatment related clinical signs were noted during daily detailed clinical observations. No findings were noted during the weekly arena observations in this study.
Five females at 120 mg/kg bw/day showed piloerection on Day 5 of dosing. This clinical sign was considered treatment related, but as it occurred on a single day of treatment only it was regarded non-adverse.
Salivation seen after dosing among all animals of the 30, 60 and 120 mg/kg bw/day dose groups starting on Day 5 of the treatment period (and in one control female on Day 12 of treatment) was considered not toxicologically relevant, taking into account the nature and mainly minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.
One female at 30 mg/kg bw/day was found dead on Day 7 post coitum. No clinical signs were observed that were associated with its death. At necropsy beginning autolysis, a soft urinary bladder wall (probably a result of autolysis) and many dark red foci on the thymus were recorded. There were no microscopic observations indicating the cause of death. At the isolated incidence observed and with the absence of a dose related trend, this death was considered an incidental occurrence rather than related to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- See Table 1.2 and 1.3 (Page 46-49) of the attached full study report -
Body weights and body weight gain were considered to have been unaffected by treatment with the test item up to 120 mg/kg bw/day.
For males, any statistical significances compared with the control group were considered not treatment related, as this was caused by relatively high control values. Males of all treated groups showed a comparable body weight gain within the normal range.
In the absence of a dose related trend no toxicological relevance was attached to the statistically significantly increased absolute body weight in females at 30 mg/kg bw/day on Day 8 of pre-mating.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- See Table 1.4 and 1.5 (Page 50-53) of the attached full study report -
Food consumption before or after correction for body weight was considered unaffected by treatment with the test item up to 120 mg/kg bw/day.
A trend was observed towards decreased food consumption in females of all treatment groups during lactation and on Days 7-14 post-coitum at 120 mg/kg bw/day. As changes compared to the concurrent control group were relatively slight (reaching no statistical significance) no toxicological significance was attached to this finding.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- See Table 1.8 (Page 63-66) of the attached full study report -
Decreased reticulocyte counts were found in males at 120 mg/kg bw/day (0.84x of control). As mean values remained within the historical control range this change was considered not toxicologically relevant.
Historical control data for Wistar Han rats; F0-animals (period 2017-2019):
Reticulocytes (109/L) – males: mean = 220.7; P5 – P95 = 163.70-289.20 (n=331).

Coagulation parameters of treated rats were considered not to have been affected by treatment. The increased mean prothrombin time (PT) in males at 120 mg/kg bw/day was caused by one individual. As mean values were within the historical control range and individual values (except for the male mentioned before) were within the same range as control, this change was considered not toxicologically relevant. In the absence of a dose related trend, decreased activated partial thromboplastin time (APTT) in males at 60 mg/kg bw/day was considered not related to treatment with the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- See Table 1.9 (Page 67-70) of the attached full study report -
Decreased total protein concentrations were found in males starting at 60 mg/kg bw/day and in females starting at 30 mg/kg bw/day (up to 0.90x and 0.93x of control for males and females, respectively, not statistically significant for females at 60 mg/kg bw/day). Mean values were below or on the lower limit of the historical control range. In males, this change was considered test item related. In the absence of a dose related trend, in females these changes were regarded not related to treatment with the test item.
Decreased albumin concentrations were measured in males starting at 60 mg/kg bw/day and in females starting at 30 mg/kg bw/day (up to 0.95x and 0.91x of control in males and females, respectively, not statistically significant for females at 60 mg/kg bw/day). As mean values remained within the historical control range (both sexes) and no dose related trend was observed (females) these changes were considered not toxicologically relevant.
Decreased cholesterol levels were found in males starting at 60 mg/kg bw/day (up to 0.76x of control, statistically significant in the high dose only). As mean values remained within the historical control range this change was considered not toxicologically relevant.
Decreased calcium levels in males were measured starting at 60 mg/kg bw/day (0.94x of control). As mean values remained within the historical control range and in the absence of a dose related trend, this change was considered not toxicologically relevant.
The change in glucose in males at 30 mg/kg bw/day was considered not related to treatment with the test item as it occurred in the absence of a dose-related trend.

Historical control data for Wistar Han rats; F0-animals (period 2017-2019):
Total protein (g/L) – males: mean = 64.2; P5 – P95 = 60.60-67.90 (n=355).
Total protein (g/L) – females: mean = 57.8; P5 – P95 = 52.80-67.90 (n=325).
Albumin (g/L) – males: mean = 32.4; P5 – P95 = 30.60-34.40 (n=355).
Albumin (g/L) – females: mean = 30.3; P5 – P95 = 27.40-36.60 (n=325).
Cholesterol (mmol/L) – males: mean = 1.95; P5 – P95 = 1.390-2.550 (n=355).
Calcium (mmol/L) – males: mean = 2.62; P5 – P95 = 2.490-2.750 (n=355).

Serum levels of total T4 in males were considered unaffected by treatment with the test item up to 120 mg/kg bw/day. Assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in the males, furthermore no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

- See Table 1.6 and 1.7 (Page 54-62) of the attached full study report -
Functional observation parameters were considered unaffected by treatment in males up to 60 mg/kg bw/day and in females up to 120 mg/kg bw/day. In males at 120 mg/kg bw/day grip strength of the hind leg was decreased (0.80x of control). This change was considered test item related.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 120 mg/kg bw/day.
Decreased total movements and ambulations in males starting at 30 mg/kg bw/day, as well as increased total movements and ambulations in females starting at 30 mg/kg bw/day were considered the result of relatively high or low control values. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period, no difference in activity between control and treated animals was observed during daily clinical observations or weekly arena observations, and no dose-related trend was observed. Altogether, the differences observed in total movements and ambulations were considered not related to treatment with the test item.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- See Table 1.11 (Page 73-76) of the attached full study report -
Test item-related higher liver weights (absolute and/or relative to body weights) were noted in males and females. For males and females, the effect on liver weight at 120 mg/kg bw/day were considered test item related. For males, the higher liver weights at 30 and 60 mg/kg bw/day were without a dose response and in absence of any microscopic findings and were therefore considered unlikely to be test item related.

Decreased absolute and relative spleen weight in females starting at 30 mg/kg bw/day (statistically significant on some occasions) was considered the result of relatively high control values and in the absence of a dose related trend or microscopic findings regarded not related to treatment with the test item.

Statistically significantly increased seminal vesicle weight (relative to body weight) in males at 120 mg/kg bw/day were considered the result of relatively low control values and in the absence of microscopic findings were regarded not related to treatment with the test item.

Statistically significantly increased absolute ovary weight in females at 30 mg/kg bw/day (1.27x of control, no significant difference was recorded for relative ovary weight) was considered not treatment related in the absence of a dose related trend.

Historical control data for Wistar Han rats; F0-animals (period 2017-2019):
Seminal vesicles (%) – males: mean = 0.384; P5 – P95 = 0.2780-0.4947 (n=737).
Spleen (g) – females: mean = 0.496; P5 – P95 = 0.3980-0.6030 (n=310).
Spleen (%) – females: mean = 0.176; P5 – P95 = 0.1445-0.2114 (n=310).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- See Table 1.10 (Page 71-72) of the attached full study report -
Test item-related enlarged liver was observed in a single female at 120 mg/kg bw/day. This correlated with higher liver weight and hepatocellular hypertrophy. The foci recorded on the glandular mucosa of the stomach in two males at 120 mg/kg bw/day were considered not toxicologically relevant in absence of correlating microscopic findings.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- See Appendix 4 (Page 191-297 of the attached full study report -
Test item and dose related microscopic findings after treatment with the test item were noted in the liver of males and females.
Hepatocellular hypertrophy (mainly periportal) was present in the liver of females treated at 60 mg/kg bw/day (minimal degree), and in males (minimal degree) and females (up to slight degree) at 120 mg/kg bw/day.
For one female at 120 mg/kg bw/day hepatocellular hypertrophy correlated with higher liver weight and with macroscopically enlarged liver.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Other effects:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by
treatment.

Details on results:

- See Table 1.12 (Page 77) of the attached full study report -
There were 2/10 couples treated at 60 mg/kg/day and 5/10 couples at 120 mg/kg/day that were not pregnant. In addition, there was one female at 60 mg/kg/day with one late resorption only. Histopathology did not reveal any changes in the reproductive organs that could explain this high incidence in reproductive failure. Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present. The males that did not fertilize the females also showed normal spermatogenesis.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to and including the highest dose tested.

Key result

Critical effects observed:

no

Conclusions:

Under the conditions of the test (OECD 422, GLP), the systemic NOAEL was determined to be at least 120 mg/kg bw/day.

Executive summary:

Introduction and method: The substance was tested in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) following GLP. The dose levels in this study were selected to be 0, 30, 60, 120 mg/kg/day, based on the results of the dose range finder in which mortality was observed at 450 mg/kg/day but no severe effects were recorded at 90 mg/kg/day. The test item and vehicle (water) were administered once daily by oral gavage 7 days a week for a minimum of 29 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50 to 57 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.

Parameters measured: Mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

Results: Mortality: One female (No. 52) at 30 mg/kg/day was found dead on Day 7 post-coitum. No clinical signs were observed that were associated with its death. At necropsy beginning autolysis, a soft urinary bladder wall (probably a result of autolysis) and many dark red foci on the thymus were recorded. There were no microscopic observations indicating the cause of death. At the isolated incidence observed and with the absence of a dose-related trend, this death was considered an incidental occurrence rather than related to treatment with the test item.

Clinical signs: No toxicologically significant changes were noted.

Functional observation tests: No toxicologically significant changes were noted.

Body weight: No toxicologically significant changes were noted.

Food consumption: No toxicologically significant changes were noted.

Haematology and clotting parameters: No effects observed.

Clinical chemistry: Decreased total protein concentrations in males starting at 60 mg/kg/day were considered non-adverse in the absence of corroborating findings.

Hormone levels: Male T4 thyroid hormone levels were not affected.

Organ weights and histopathology: Relationships were suspected between enlarged liver, increased liver weight and hepatocellular hypertrophy. The (mainly periportal) hepatocellular hypertrophy in females at 60 and 120 mg/kg/day and males at 120 mg/kg/day was considered non-adverse at the current severities (minimal to slight) and in the absence of any degenerative findings. Therefore, the higher liver weights (up to 19%) in males and females were considered non-adverse.

Conclusion: In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental NOAEL is at least 120 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test

Version / remarks:

2000

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-propionaldehyde

EC Number:

251-717-2

EC Name:

6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-propionaldehyde

Cas Number:

33885-51-7

Molecular formula:

C12H18O

IUPAC Name:

3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)propanal

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 13-14 weeks
- Weight at study initiation: 195 - 249 g (females)
- Fasting period before study: No (except for males which were fasted overnight with a
maximum of 24 hours before sacrifice; females were not fasted)
- Housing: On arrival and following the pretest 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). After mating, 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). During locomotor activity monitoring, animals were housed individually in a Hi-temp poly
carbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment,
bedding material, food and water. The cages contained appropriate bedding.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad
libitum)
- Water: Municipal tap water, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants.
Periodic analysis of the water was performed. It is considered that there were no known contaminants in the feed and in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 45-56
- Air changes (per hr): 10 or more air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 March 2020 To: 01 May 2020

Administration / exposure

Route of administration:

oral: gavage

Details on exposure:

The test item was administered as received. An adequate amount of the test item was dispensed into daily aliquots, which were stored the same as for the bulk test item until use. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the test item (0.9584 (D25/25)). No correction was made for the purity/composition of the test item.
The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. Dose volumes ≤ 50 µL were administered using a digital syringe and dose volumes > 50 µL were given with a plastic feeding tube connected to an appropriately graded syringe.
A dose control system was used as additional check to verify the dosing procedure according to
Standard Operating Procedures.
Dose volumes were 0.0313, 0.0626, 0.125 mL/kg bw for the low, mid and high dose groups, respectively. The control animals received 0.125 mL water/ kg bw.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

During the mating phase, males and females were cohabitated on a 1:1 basis, 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.

Duration of treatment / exposure:

Females that delivered were treated for 50 to 57 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 treated for 41 to 43 days (except for one female, which failed to delivery but died spontaneously after 25 days of treatment.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

Frequency of treatment:

Once daily, 7 days/week

Duration of test:

Maximum 58 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Low dose group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Mid dose group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

High dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of a 14-day Dose Range Finder with oral
administration of Pino Acetaldehyde in rats (Test Facility Reference No. 20137515) in which mortality was observed at 450 mg/kg bw/day but no severe effects were recorded at 90 mg/kg bw/day. In this study significant treatment related changes were recorded for sperm parameters at 450 mg/kg bw/day (reduced mean motile sperm, mean progressive motility, mean number of cells with normal morphology and mean number of cells with coiled tail, and increased mean number of cells with detached head and mean number of cells with abnormal neck. Epididymal sperm count was not considered affected by treatment. These findings were not recorded at 90 mg/kg bw/day.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for general health/mortality and moribundity
- The circumstances of any death were recorded in detail.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
- During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals (no peak effect of occurrence of clinical signs was observed in the dose range finder)
- The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.
- Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. The terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION:
Yes
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION: Yes
- Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

FUNCTIONAL TESTS:
Functional tests were performed on 5 females/group during the last week of lactation (i.e. PND 6-13), These tests were performed after dosing, after completion of clinical observations (including arena observation, if applicable).
The following tests were performed:
- Hearing ability;
- Pupillary reflex;
- Static righting reflex;
- Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal;
- Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

ESTROUS CYCLE DETERMINATION
- 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 estrous.

Ovaries and uterine content:

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females.
In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

Blood sampling:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: 5/sex/group
- Parameters as specified in the OECD guideline were included (coagulation parameters (prothrombin time and activated partial thromboplastin time) were included.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: 5/sex/group
- Parameters as specified in the OECD guideline were included

Fetal examinations:

Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally). Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day.
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.

All remaining pups were euthanized on PND 14-16. 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.

Pups were observed daily for general health/mortality. The number of live and dead pups was 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. 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.

Measurement of total T4 hormone was conducted for PND 14-16 pups. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16

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% 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 3 observations.
The following pairwise comparisons were made:
Low dose group vs. control group, mid dose group vs. control group, high dose group vs. control
group.
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
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.

Indices:

Mating index (%): (Number of females mated/ Number of females paired) x 100;
Precoital time: Number of days between initiation of cohabitation and confirmation of mating;
Fertility index (%): (Number of pregnant females/ Number of females mated) x 100;
Gestation index (%): (Number of females with living pups on Day 1/ Number of pregnant females) x 100;
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition;
Post-implantation survival index (%): (Total number of offspring born/ Total number of uterine implantation sites) x 100;
Live birth index (%): (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100;
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/ Number of live pups at First Litter Check) x100;
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/ Number of live pups at First Litter Check) x 100;
Viability index (%): (Number of live offspring on Day 4 before culling/ Number live offspring on Day 1 after littering) x 100;
Lactation index (%): (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- See Table 1.11 (Page 45) of the attached full study report under IUCLID section 7.5.1-
No adverse treatment related clinical signs were noted during daily detailed clinical observations. No findings were noted during the weekly arena observations in this study. Five females at 120 mg/kg bw/day showed piloerection on Day 5 of dosing. This clinical sign was considered treatment related, but as it occurred on a single day of treatment only it was regarded non-adverse. Salivation seen after dosing among all animals of the 30, 60 and 120 mg/kg bw/day dose groups starting on Day 5 of the treatment period (and in one control female on Day 12 of treatment) was considered not toxicologically relevant, taking into account the nature and mainly minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.
One female at 30 mg/kg bw/day was found dead on Day 7 post coitum. No clinical signs were observed that were associated with its death. At necropsy beginning autolysis, a soft urinary bladder wall (probably a result of autolysis) and many dark red foci on the thymus were recorded. There were no microscopic observations indicating the cause of death. At the isolated incidence observed and with the absence of a dose related trend, this death was considered an incidental occurrence rather than related to treatment with the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- See Table 1.2 and 1.3 (Page 47, 49) of the attached full study report under IUCLID section 7.5.1-
Body weights and body weight gain were considered to have been unaffected by treatment with the test item up to 120 mg/kg bw/day. In the absence of a dose related trend no toxicological relevance was attached to the statistically significantly increased absolute body weight in females at 30 mg/kg bw/day on Day 8 of pre mating.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- See Table 1.4 and 1.5 (Page 51, 53) of the attached full study report under IUCLID section 7.5.1 -
Food consumption before or after correction for body weight was considered unaffected by treatment with the test item up to 120 mg/kg bw/day. A trend was observed towards decreased food consumption in females of all treatment groups during lactation and on Days 7-14 post-coitum at 120 mg/kg bw/day. As changes compared to the concurrent control group were relatively slight (reaching no statistical significance) no toxicological significance was attached to this finding.

Haematological findings:

no effects observed

Description (incidence and severity):

- See Table 1.8 (Page 65-66) of the attached full study report under IUCLID section 7.5.1 -
No effects were observed in females.

Description (incidence and severity):

- See Table 1.9 (Page 69-70) of the attached full study report under IUCLID section 7.5.1-
Decreased total protein concentrations were found in females starting at 30 mg/kg bw/day (up to 0.90x and 0.93x of controls), not statistically significant for females at 60 mg/kg bw/day). Mean values were below or on the lower limit of the historical control range.
Decreased albumin concentrations were measured in females starting at 30 mg/kg bw/day (up to 0.91x of control, not statistically significant for females at 60 mg/kg bw/day). As mean values remained within the historical control range (both sexes) and no dose related trend was observed (females) these changes were considered not toxicologically relevant.
Historical control data for Wistar Han rats; F0-animals (period 2017-2019):
Total protein (g/L) – females: mean = 57.8; P5 – P95 = 52.80-67.90 (n=325).
Albumin (g/L) – females: mean = 30.3; P5 – P95 = 27.40-36.60 (n=325).

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- See Table 1.6 and 1.7 (Page 54, 59-62) of the attached full study report under IUCLID section 7.5.1-
Functional observation parameters were considered unaffected by treatment in females up to 120 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- See Table 1.11 (Page 75-76) of the attached full study report under IUCLID section 7.5.1-
Test item-related higher liver weights (absolute and/or relative to body weights) were noted in females. For females, the effect on liver weight at 120 mg/kg bw/day were considered
test item related.
Decreased absolute and relative spleen weight in females starting at 30 mg/kg bw/day (statistically significant on some occasions) was considered the result of relatively high control values and in the absence of a dose related trend or microscopic findings regarded not related to treatment with the test item.
Statistically significantly increased absolute ovary weight in females at 30 mg/kg bw/day (1.27x
of control, no significant difference was recorded for relative ovary weight) was considered not tr
eatment related in the absence of a dose related trend.
Historical control data for Wistar Han rats; F0-animals (period 2017-2019):
Spleen (g) – females: mean = 0.496; P5 – P95 = 0.3980-0.6030 (n=310).
Spleen (%) – females: mean = 0.176; P5 – P95 = 0.1445-0.2114 (n=310).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- See Table 1.10 (Page 71-72) of the attached full study report -
Test item-related enlarged liver was observed in a single female at 120 mg/kg bw/day. This correlated with higher liver weight and hepatocellular hypertrophy. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- See Appendix 4 (Page 191-297) of the attached full study report under IUCLID section 7.5.1 -
Test item and dose related microscopic findings after treatment with the test item were noted in the liver of females.
Hepatocellular hypertrophy (mainly periportal) was present in the liver of females treated at 60 mg/kg bw/day (minimal degree) and females (up to slight degree) at 120 mg/
kg bw/day.
For one female at 120 mg/kg bw/day hepatocellular hypertrophy correlated with higher liver weight and with macroscopically enlarged liver.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

- See Appendix 4 (Page 191-297) of the attached full study report under IUCLID section 7.5.1 -
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.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- See Table 1.14 (Page 79) and 1.18 (Page 81) of the attached full study report under IUCLID section 7.5.1 -
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment up to 60 mg/kg bw/day.
Post-implantation survival index was 91, 97, and 93% for the control, 30, and 60 mg/kg bw/day groups, respectively. The high dose group is not included in the summary based on the fact that the number of pregnant dams was too low.
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment up to 60 mg/kg bw/day.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

- See Table 1.12 (Page 77) of the attached full study report under IUCLID section 7.5.1 -
There was one female at 60 mg/kg bw/day with one late resorption only. This was considered an incidental finding and not related to exposure to the test item.

Dead fetuses:

no effects observed

Description (incidence and severity):

- See Table 1.15 (Page 80) of the attached full study report under IUCLID section 7.5.1 -
Litter size was considered not affected by treatment at 30 and 60 mg/kg bw/day.
Live litter sizes were 11.7, 12.1, and 12.0 living pups/litter for the control, 30, and 60 mg/kg bw/day groups, respectively.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

- See Table 1.12 (Page 77) of the attached full study report under IUCLID section 7.5.1 -
Gestation index and duration of gestation were considered not to be affected by treatment up to 60 mg/kg bw/day.
Except for one female at 60 mg/kg bw/day, all pregnant females had live offspring. The gestation indices were 100% for the control and 30 mg/kg bw/day groups and 88% for the 60 mg/kg bw/day group. As one female without live offspring is within the normal range, this difference was considered not toxicologically relevant.

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed up to and including the highest dose tested (120 mg/kg bw/day)

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

- See Table 1.16 (Page 82) of the attached full study report under IUCLID section 7.5.1 -
Body weights of pups were considered not to be affected by treatment at 30 mg/kg bw/day.
At 60 mg/kg bw/day pup body weights (both sexes) were decreased compared with control from PND 1 onwards. Mean body weights of male and female pups together were decreased with 15.6% on PND 1, slightly recovering to 9.3% on PND 13. This difference was considered treatment related.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- See Table 1.14 and 1.15 (Page 80-81) of the attached full study report under IUCLID section 7.5.1 -
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment at 30 and 60 mg/kg bw/day.
The live birth indices were 100, 100, and 99% for the control, 30, and 60 mg/kg bw/day groups, respectively.
One pup at 60 mg/kg bw/daywas found dead at first litter check. At this single occurrence, it was considered not toxicologically relevant.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- See Table 1.14 (Page 80) of the attached full study report under IUCLID section 7.5.1 -
Sex ratio was considered not to be affected by treatment at 30 and 60 mg/kg bw/day.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

- See Table 1.17 and 1.18 (Page 83-84) of the attached full study report under IUCLID section 7.5.1 -
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment at 30 and 60 mg/kg bw/day.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

- See Table 1.15 (Page 81) of the attached full study report under IUCLID section 7.5.1 -
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment at 30 and 60 mg/kg bw/day.
Viability indices were 100% for the control and 30 mg/kg bw/day groups and 96% for the 60 mg/kg bw/day group.
Three pups at 60 mg/kg bw/day were found missing on PND 2 or 4 (each from a different litter).
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment at 30 and 60 mg/kg bw/day.
The lactation indices were 99, 97, and 100% for the control, 30, and 60 mg/kg bw/day groups, respectively.
One pup of the control group was found dead on PND 10. Two pups at 30 mg/kg bw/day were found missing on PND 7 or 10 (each from a different litter). Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

External malformations:

no effects observed

Description (incidence and severity):

- See Table 2.23 (Page 168-185) of the attached full study report under IUCLID section 7.5.1 -
No macroscopic findings were noted in pups at all dose groups surviving until scheduled necropsy.

Other effects:

no effects observed

Description (incidence and severity):

- See Table 2.23 (Page 168-185) of the attached full study report under IUCLID section 7.5.1 -
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 considered not to be toxicologically relevant.

Treatment at 30 and 60 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Serum levels of total T4 in male and female PND 14-16 pups were considered not to be affected by treatment at 30 and 60 mg/kg bw/day.

Details on embryotoxic / teratogenic effects:

As three females at 60 mg/kg bw/day and 5 female at 120 mg/kg bw/day were not pregnant or had a late resorption only, developmental data is available of only 7 and 5 females in the mid and high dose group, respectively. The data at 120 mg/kg bw/day was considered too limited for a toxicological evaluation. Therefore, developmental results on the high dose group are not summarized, but included below.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at 60 mg/kg bw/day.

Remarks on result:

other: Developmental results were not evaluated at 120 mg/kg bw/day due to the limited number of litters in this group.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD 422, GLP), the systemic NOAEL was determined to be at least 120 mg/kg bw/day. The developmental NOAEL is >= 60 mg/kg bw (the high dose was not evaluated as there were insufficient litters).

Executive summary:

Introduction and method: The substance was tested in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) following GLP. The dose levels in this study were selected to be 0, 30, 60, 120 mg/kg/day, based on the results of the Dose Range Finder in which mortality was observed at 450 mg/kg/day but no severe effects were recorded at 90 mg/kg/day. The test item and vehicle were administered once daily by oral gavage 7 days a week for a minimum of 29 days. Females that delivered were treated for 50 to 57 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.

Parameters measured: Mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

Results: Mortality: One female (No. 52) at 30 mg/kg/day was found dead on Day 7 post-coitum. No clinical signs were observed that were associated with its death. At necropsy beginning autolysis, a soft urinary bladder wall (probably a result of autolysis) and many dark red foci on the thymus were recorded. There were no microscopic observations indicating the cause of death. At the isolated incidence observed and with the absence of a dose-related trend, this death was considered an incidental occurrence rather than related to treatment with the test item.

Clinical signs: No toxicologically significant changes were noted.

Functional observation tests: No toxicologically significant changes were noted.

Body weight: No toxicologically significant changes were noted.

Food consumption: No toxicologically significant changes were noted.

Haematology and clotting parameters: No effects observed.

Clinical chemistry: No toxicologically significant changes were noted

Organ weights and histopathology: Relationships were suspected between enlarged liver, increased liver weight and hepatocellular hypertrophy. The (mainly periportal) hepatocellular hypertrophy in females at 60 and 120 mg/kg/daywas considered non-adverse at the current severities (minimal to slight) and in the absence of any degenerative findings. Therefore, the higher liver weights (up to 19%) in were considered non-adverse.

Fertility: Fertility index was adversely affected at 120 mg/kg/day, with a fertility index of 50%. No reproduction toxicity was observed up to 60 mg/kg/day. The two non-pregnancies in the mid dose group were considered to be within normal ranges of biological variation. In this study, no treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating index, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental toxicity: No adverse developmental toxicity was observed at 30 and 60 mg/kg/day. Developmental data was available of only 7 and 5 females in the mid and high dose group, respectively. One animal in the mid dose group was pregnant but fetuses were resorbed. As the other litters in the mid dose group showed consistent results, developmental parameters could be evaluated despite a slightly low number of litters in this group (< 8 litters). The 5 litters for the high dose group were considered too limited for a toxicological evaluation. At 60 mg/kg/day pup body weights (both sexes) were decreased compared with control from PND 1 onwards. As body weights partly recovered from PND 1 up to PND 13 (from 15.6% to 9.3%) this difference was considered non-adverse. No toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

Conclusion: In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse effect level (NOAEL) were established within the current study: Parental NOAEL is at least 120 mg/kg/day. The reproduction NOAEL is 60 mg/kg/day based on decreased fertility index at 120 mg/kg/day. The developmental NOAEL is at least 60 mg/kg/day as the highest dose could not be evaluated as there were only 5 litters.