3-p-cumenyl-2-methylpropionaldehyde

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

OECD 415, OECD 408, 28 day in rats, 14 -day in rabbits, in vitro metabolism studies.

Overall the data can be summarised as follows:

- Cyclamen Aldehyde has effects on rat spermatogenesis at 75 mg/kg/day;

- Cyclamen Aldehyde has no effects on rabbit spermatogenesis at levels of 300 mg/kg/day;

- The presence of the toxic metabolite 4 -isopropyl benzoic acid conjugate with CoA (iPBA-CoA), strongly correlated to male rat reproductive toxicity for a number of structurally related compounds, has been confirmed in rat plasma, hepatocytes and testes, but not in plated rabbits and humans hepatocytes;

- Species specific metabolic fate linked to CoA toxicity in male rats can be postulated based on analytical data in vitro and in vivo in the liver, and in male reproductive tissue in vivo;

- There is strong evidence that this species specific metabolic fate in the rat is not relevant to the rabbit and humans.

Cyclamen aldehyde did not affect female rat fertility parameters.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Aug 2009 - 26 May 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

The requirements of the Organisation for Economic Co-operation and Development Guidelines for Testing of Chemicals No. 415 were used as the basis for study design


- Basis for dose level selection
The reproductive effects of cyclamen aldehyde in corn oil were evaluated in TIF00041. In that study, dosages of 0 (Vehicle), 25, 75, and 150 mg/kg/day were administered orally (via gavage) to P generation male and female rats at a volume of 4 mL/kg. Treated rats were mated with untreated cohorts of male and female rats. Male rats were given the vehicle or test substance once daily beginning 14 days before cohabitation, through cohabitation, and continuing through the day before sacrifice, while female rats were given the vehicle or test substance once daily beginning 14 days before cohabitation, through cohabitation, and continuing through day 24 of presumed gestation (DG 24) (rats that did not deliver a litter), day 4 postpartum (DL 4) (rats that delivered a litter), or
DS 44 (rats with no confirmed date of mating). In treated male rats, reproductive organ weights were reduced in the 75 and 150 mg/kg/day dosage group. Adverse effects on sperm analyses and histopathological changes to the epididymides were observed in the 150 mg/kg/day dosage group.

In treated female rats, reduced gestational body weights were observed at the 150 mg/kg/day dosage. At 75 and 150 mg/kg/day, reduced pup body weights were observed. In untreated females mated to the treated males (Cohort 2), a reduced number of implantation sites and a reduced fertility index were observed at 150 mg/kg/day. The dosages of 0 (Vehicle), 25, 75, and 150 mg/kg/day were selected for this study.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Crl:CD(SD) rat was selected as the Test System because of known response to toxic effects on reproductive capacity and history of use as a rodent species in these evaluations.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Twenty-five Crl:CD(SD) strain rats/sex/dose were administered the test material at dosages of 0, 25, 75 or 150 mg/kg/day in corn oil. Dose volume was 4 ml/kg which was adjusted daily on the basis of the individual bodyweights.
These treated male and treated female rats were cohabitated (for a maximum of 21 days) with untreated cohort male or female rats. Treated P generation male rats were 38 days old upon arrival and weighed 131-166 grams at study start. The untreated P generation male rats were 122 days old upon arrival and weighed 437-550 grams at study start. Treated P generation female rats were 66 days old upon arrival and weighed 218-285 grams at study start. Untreated P generation female rats were 66 days old upon arrival and weighed 224-311 grams at study start.

Conditions
The study rooms were maintained under conditions of positive airflow relative to a hallway and independently supplied with a minimum of 10 changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters. Room temperature was maintained at 64 F to 79 F (18 C to 26 C); relative humidity was targeted at 30% to 70 %. P generation rats were housed individually in stainless steel, wire-bottomed cages, except during the cohabitation and postpartum periods. During cohabitation, each pair of male and female rats was housed in the male rat's cage. Beginning no later than DG 20, P generation female rats were individually housed in nesting boxes until they either naturally delivered litters or were sacrificed on DG 25. Each dam and delivered litter were housed in a common nesting box during the postpartum period. After weaning, F1 generation pups selected for continued evaluation were individually housed in stainless steel, wire-bottomed cages. A 12 hour light/dark cycle was maintained. Cage pan liners were changed at least 3 times per week. Cages were changed approximately every other week. Bedding was changed as often as necessary to keep the rats dry and clean. Rats were given ad libitum access to Certified Rodent Diet #5002 meal in individual feeders. Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad libitum from an automatic watering access system and/or individual water bottles attached to the cages. Chlorine was added to the processed water as a bacteriostat. Bed-o'-cobs bedding was used as the nesting material. Chewable Nylabones were supplied to all rats during the study.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Male P generation rats were gavaged once daily beginning 83 days prior to cohabitation, through cohabitation, continuing through the day before sacrifice. Female P generation rats were gavaged once daily beginning 14 days before cohabitation, through cohabitation and DG 25 (rats that did not deliver) or day 22 postpartum (rats that delivered a litter). F1 generation rats were not directly dosed but may have been exposed to the test material in utero during gestation and through maternal milk postpartum.

Details on mating procedure:

Treated or untreated female rats wtih spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at DG 0 and assigned to individual housing. Treated female rats not mated within the first 14 days of cohabitation were assigned alternate male rats that had mated (same dosage group) and remained in cohabitation for a maximum of 7 additional days. Treated male rats that did not mate an untreated female rat within the first 14 days of cohabitation were assigned an alternate untreated female rat and remained in cohabitation for a maximum of 7 additional days. Treated or untreated female rats not mated after completing of the 21 day cohabitation period were considered at DG 0 on the last day of cohabitation and assigned to individual housing. Three untreated female rats not mated with a treated male rat within the first 14 days of cohabitation were considered DG 0. Day 1 of lactation (postpartum) was defined as the day of birth and was also the first day of which all pups in a litter were individually weighed. All F1 generation rats were weaned on PPD 22, based on observed growth and viability of the pups. At weaning, 25 male and 25 female pups per group from treated dams (mated with untreated male rats) and 25 male and 25 female pups per group from untreated dams (mated withtreated male rats) were selected resulting in a total of 350 F1 generation rats (175 per sex) chosen for continued evaluation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration and homogeneity were assessed for all test article groups while only concentration was verified for the vehicle group (Group I). Quadruplicate samples were taken from the top, middle and bottom of each concentration on the first day of preparation. Two samples from each quadruplicate set were shipped for analysis; the remaining samples were retained at the Testing Facility as backup samples. Quadruplicate samples were taken from each concentration at the mid-point of the study period and on the last day of preparation. Two samples from each quadruplicate set were shipped to Charles River Laboratories Preclinical Services, Ohio for analysis; the remaining samples were retained as backup samples. Backup samples were stored at room temperature and protected from light. A quadruplicate set of samples was collected from the lowest concentration prepared on the first day of preparation for verification of formulation stability. All stability samples were shipped for analysis and storage as backups.

Duration of treatment / exposure:

Described at Details on exposure section.

Frequency of treatment:

Once daily.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Concentration (mg/mL): 0
Dosage Volume (mL/kg): 4

Dose / conc.:

25 mg/kg bw/day

Remarks:

Concentration (mg/mL): 6.25
Dosage Volume (mL/kg): 4

Dose / conc.:

75 mg/kg bw/day

Remarks:

Concentration (mg/mL): 18.75
Dosage Volume (mL/kg): 4

Dose / conc.:

150 mg/kg bw/day

Remarks:

Concentration (mg/mL): 37.5
Dosage Volume (mL/kg): 4

No. of animals per sex per dose:

Twenty-five Crl:CD(SD) strain rats/sex/dose were administered the test material at dosages of 0, 25, 75 or 150 mg/kg/day in corn oil. Dose volume was 4 ml/kg which was adjusted daily on the basis of the individual bodyweights.

These treated male and treated female rats were cohabitated (for a maximum of 21 days) with untreated cohort male or female rats. Treated P generation male rats were 38 days old upon arrival and weighed 131-166 grams at study start. The untreated P generation male rats were 122 days old upon arrival and weighed 437-550 grams at study start. Treated P generation female rats were 66 days old upon arrival and weighed 218-285 grams at study start. Untreated P generation female rats were 66 days old upon arrival and weighed 224-311 grams at study start.

Control animals:

yes, concurrent vehicle

Positive control:

No data

Parental animals: Observations and examinations:

The following parameters were evaluated in the P generation rats (treated or untreated): viability, clinical observations, body weights, feed consumption, estrous cycling, mating and fertility, natural delivery and litter observations, sperm assessments (motility and concentration), organ weights, histopathology and/or necropsy observations.

Oestrous cyclicity (parental animals):

Estrous cycling was evaluated daily by examination of vaginal cytology beginning 28 days (treated female rats) or 14 days (untreated female rats) before scheduled cohabitation and continuing through cohabitation until mating was confirmed or until the end of the cohabitation period was reached.

Sperm parameters (parental animals):

To assess the potential toxicity of the test material in P generation male rats on the male reproductive system, organ weights, sperm evaluation and histopathology were evaluated. Sperm concentration and motility were evaluated.

Litter observations:

Treated rats were observed for clinical signs, abortions, premature deliveries and deaths daily before dosage administration, between one and two hours following dosage administration and at the end of the normal working day. Untreated rats were observed for clinical signs, abortions, premature deliveries and deaths weekly during the precohabitation and cohabitation periods, on DG 0, twice weekly during the gestation period and on days 1, 5, 8, 11, 15, 18 and 22 postpartum. In addition, treated and untreated female rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter size (all pups delivered) and pup viability at birth. Treated and untreated dams and their respective litters were observed for maternal behaviour on days 1, 5, 8, 14, 18 and 22 postpartum.
. F1 generation pups were individually weighed and litters were examined after delivery to identify the number and sex of pups, stillbirths, live births and gross alterations. Anogenital distance was measured for all live F1 generation pups on days 1 and 22 postpartum. Nipple eruption was evaluated for all live F1 generation pups once on day 12 postpartum. F1 generation litters were observed at least twice daily for viability. The pups in each litter were counted once daily. Clinical observations were recorded once daily during he preweaning period. Pup body weights were recorded on days 1 (birth), 5, 8, 11, 15, 18 and 22 postpartum, and at least weekly during the postweaning period and on the day of sacrifice. During the postweaning period, rats were observed twice daily for viability and once daily for clinical observations and/or general appearance. Body weights and feed consumption values were recorded weekly during the postweaning period. Female F1 generation rats were evaluated for the age of vaginal patency, beginning on day 28 postpartum and continuing until this developmental parameter is achieved, or until day 40 postpartum.

Postmortem examinations (parental animals):

The body weight was recorded for each rat on the day the criterion was achieved/recorded. A terminal body weight was also recorded. Treated P generation male rats that died or were sacrificed before scheduled termination were examined for the cause of death or condition on the day the observation was made. The rats were examined for gross lesions and a complete necropsy was performed. Tissues were retained and/or microscopically evaluated, but not weighed. Surviving treated and untreated P generation male rats were sacrificed by CO2 asphyxiation after completion of the cohabitation period. Treated and untreated female rats that delivered a litter and their respective pups not selected for continued evaluation were sacrificed on day 22 of presumed lactation (DL 22). Female rats that did not deliver a litter were sacrificed on day 25 of gestation (DG 25). Male and female F1 generation rats weaned and selected for continued evaluation were sacrificed by CO2 asphyxiation on day 57, 58 or 60 postpartum. All surviving treated P generation male and female rats were subject to a complete necropsy examination which included:
evaluation of the carcass and musculoskeletal system;
all external surfaces and orifices;
cranial cavity and external surfaces of the brain and spinal column;
the nasal cavity and neck with associated organs and tissues;
and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
To assess the potential toxicity of the test material in P generation male rats on the male reproductive system, organ weights, sperm evaluation and histopathology were evaluated. Sperm concentration and motility were evaluated. Treated dams with no surviving pus were sacrificed after the last pup was found dead or missing and presumed cannibalized. The number and distribution of implantation sites were recorded. Uteri of apparently nonpregnant rats were examined while being pressed between glass plates to confirm the absence of implantation sites, and retained in 10% Neutral buffered formalin for microscopic evaluation. Tissues identified for microsocpic evalution were embedded in paraffin, sectioned, mounted on glass slides and stained with hematoxylin and eosin. Histopathological evaluation was performed by a board-certified veterinary pathologist. Untreated P generation male rats were sacrificed via CO2 asphyxiation after completion of the cohabitation period and carcasses were discarded without evaluation. Untreated P generation female rats were sacrificed via CO2 asphyxiation after completion of the 22 day postpartum period. The number and distribution of implantation sites were recorded. Untreated female rats that did not deliver a litter were sacrificed on DG 25 and examined for pregnancy status. Uteri of apparently non-pregnant rats were examined to confirm the absence of implantation sites. Female rats without a confirmed mating date that did not deliver a litter were sacrificed on an estimated DG 25. The carcasses of the untreated female rats were discarded without further evaluation.

Postmortem examinations (offspring):

Unscheduled deaths of F1 generation pups were evaluated as follows: pups that died before examination of the litter for pup viability were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sunk were identified as stillborn; pups with lungs that floated were identified as liveborn and to have died shortly after birth. Pups with gross lesions were preserved in Bouin's solution for possible future evlaution. Pups that died or were sacrified before schedule termination were examined for gross lesions and the cuase of death or condition on the day the observation was made. Pups found on PPDs 2 to 5 were preserved in Bouin's solution for possible future evaluation. Pups found on days 6 to 22 postpartum were preserved in neutral buffered 10% formalin for possible future evaluation. Pups that died or were sacrified before scheduled termination from days 5 through 22 postpartum were examined for gross lesions and the cause of death or condition on the day the observation is made. All pups selected for continued evaluation were sacrified by CO2 asphyxiation on PPD 22 and examined for gross lesions; gross lesions were preserved in 10% NBF for possible future histopathological evaluation. Male and female F1 generation rats were sacrificed by CO2 asphyxiation on days 57, 58 and 60 postpartum. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Gross lesions were preserved in 10% NBF. Tissues selected were weighed and preserved in 10% NBF. Statistical analyses were conducted on data.

Statistics:

Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.
Continuous data (e.g., body weights, feed consumption values, organ weights, and percent mortality per litter) were analyzed under the Parametric heading of the schematic. Bartlett’s Test of Homogeneity of Variances was used to estimate the probability that the dosage groups have different variances. A non-significant result (p> 0.001) indicated that an assumption of homogeneity of variance was not inappropriate, and the data were compared using the Analysis of Variance. If that test was significant (p≤ 0.05), the groups given the test substance was compared with the control group using Dunnett’s Test. If Bartlett’s Test was significant (p≤ 0.001), the Analysis of Variance Test was not appropriate, and the data were analyzed as described under the Nonparametric heading of the schematic. When 75% or fewer of the scores in all the groups were tied, the Kruskal-Wallis Test was used to analyze the data, and in the event of a significant result (p≤ 0.05), Dunn’s Test was used to compare the groups given the test substance with the control group. When more than 75% of the scores in any dosage group were tied, Fisher’s Exact Test was used to compare the proportion of ties in the dosage group. Anogenital distances for F1 generation pups (sired by untreated male rats mated with treated female rats) were analyzed as described above and were then subjected to an Analysis of Covariance (ANCOVA) using mean litter weight as the covariate.
Count data were evaluated using the procedures described above for the Kruskal-Wallis Test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The number of male rats with slight or moderate excess salivation was significantly increased (p≤0.01) in the 150 mg/kg/day dosage group, in comparison to the vehicle control group value. This observation occurred intermittently during the dosage period, and was not considered an adverse effect of cyclamen aldehyde. All other clinical observations were considered unrelated to cyclamen aldehyde because: 1) the incidences were not dose related; 2) the observations were common for this species and strain of laboratory rat; and/or 3) the number of rats affected was not statistically significant when compared to the vehicle control group.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

There were no treatment-related deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES:
Reductions in body weight gain occurred in the 150 mg/kg/day dosage group intermittently during the dosage period prior to cohabitation, with statistically significant reductions (p≤0.01) occurring on DSs 50 to 57 and DSs 64 to 71, in comparison to the vehicle control group values. Body weight gains were also reduced in the 150 mg/kg/day dosage group on DSs 84 to 92 and at each tabulated interval between DS 99 and DS 134, in comparison to the vehicle control group values. Reflecting lower weight gains that occurred intermittently during the dosage period, the overall body weight gain in the 150 mg/kg/day dosage group was significantly reduced (p≤0.05) on DSs 1 to 134 a nd DS 1 to termination. Body weight gains in the 25, 75 and 150 mg/kg/day dosage groups were 98%, 95% and 92% of the vehicle control group value, respectively, on DSs 1 to 84 (the dosage period prior to cohabitation) and 100%, 96% and 90% of the vehicle control group value, respectively, for the cumulative dosage period (DSs 1 to 134).

The average body weight was minimally reduced (5% to 7% less than vehicle controls) in the 150 mg/kg/day dosage group beginning on DS 71 and continuing until scheduled euthanasia. These reductions reached statistical significance (p≤0.05) only on DS 120 when compared to the vehicle control group value. The average body weight on DS 134 % as 100%, 97% and 93% of the vehicle control group value in the three respective cyclamen aldehyde-treated groups.

FEMALES:
Precohabitation
During the first week of the dosage period (DSs 1 to 8), body weight gains were reduced at 75 mg/kg/day (64% of the vehicle control group value) and signifcantly reduced (p≤0.01) at 150 mg/kg/day (44% of the vehicle control group value), as compared to the vehicle controls. These reductions were transient and did not persist during the second week of the dosage period (DSs 8 to 15). Despite the rebound during the second week of the dosage period, body weight gains in the 75 and 150 mg/kg/day dosage groups remained reduced (75 mg/kg/day) or significantly reduced (p≤0.01 at 150 mg/kg/day) for the entire premating dosage period (DSs 1 to 15), as compared to the vehicle control group value. Body weights and body weight gains during the premating period were unaffected by the 25 mg/kg/day dosage of cyclamen aldehyde. The average body weight on DS 15 was 98%, 98% and 97% of the vehicle control group value in the 25, 75 and 150 mg/kg/day dosage groups, respectively.

Gestation
At 150 mg/kg/day, body weight gains remained reduced (by 5% to 21%) at each tabulated interval within the gestation dosage period relative to the vehicle control group values. Although the reductions within the gestation period did not reach statistical significance, the cumulative maternal body weight gains (DGs 0 to 21) were significantly reduced (p≤0.01), as compared to the vehicle control group value. Maternal body weight gains in the 25, 75 and 150 mg/kg/day dosage groups were 102%, 95% and 85% of the vehicle control group value, respectively, on DGs 0 to 21. The average maternal body weight on DG 18 and DG 21 was significantly reduced (p≤0.01) in the 150 mg/kg/day dosage group, in comparison to the vehicle control group values. The average body weight on DG 21 was 99%, 97% and 92% of the vehicle control group value in the 25, 75 and 150 mg/kg/day dosage groups, respectively. Body weights and body weight gains during the gestation period were unaffected by the 25 and 75 mg/kg/day dosages of cyclamen aldehyde.

Lactation
Body weight losses were observed in the 25, 75 and 150 mg/kg/day dosage groups at the beginning of the lactation period (DLs 1 to 5), compared to gains in the vehicle control group during this same period. The losses in body weight were more pronounced at 150 mg/kg/day, and also reached statistical significance (p≤0.01 on DLs 1 to 5) when compared to the vehicle control group value. Body weight gains rebounded in each cyclamen aldehyde-treated during the next tabulated interval (DLs 5 to 8), but the weight gains at 150 mg/kg/day remained significantly reduced (p≤0.05) in comparison to the vehicle control group value. Thereafter, body weight gains were comparable to or significantly increased (p≤0.05 or p≤0.01) when compared to the vehicle control group values.
The average maternal body weight was significantly reduced (p≤0.01) in the 150 mg/kg/day dosage group on DLs 5 through 11, in comparison to the vehicle control group values. Maternal body weight gains in the 25, 75 and 150 mg/kg/day dosage groups were 108%, 144% and 124% of the vehicle control group value, respectively, on DLs 1 to 22. The average body weight on DL 22 was 100%, 103% and 101% of the vehicle control group value in the 25, 75 and 150 mg/kg/day dosage groups, respectively. The statistically significant reduction (p≤0.05) in the average maternal body weight that occurred in the 75 mg/kg/day dosage group on DL 8 was considered unrelated to cyclamen aldehyde because the reduction was transient and did not persist. In addition, the statistically significant increase (p≤0.01) in body weight gains that occurred at 75 mg/kg/day on DLs 8 to 11 and DLs 1 to 22 were considered unrelated to cyclamen aldehyde because the increases were not dose related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MALES:
Absolute and relative feed consumption values were unaffected by dosages of cyclamen aldehyde as high as 150 mg/kg/day. Absolute feed consumption values in the 25, 75 and 150 mg/kg/day dosage groups were 100% of the vehicle control group value on DSs 1 to 84 (the dosage period prior to cohabitation) and 102%, 102% and 104% of the vehicle control group value, respectively, on DSs 106 to 134. Relative feed consumption values in the 25, 75 and 150 mg/kg/day dosage groups were 101%, 102% and 103% of the vehicle control group value, respectively, on DSs 1 to 84, and 102%, 104% and 111% of the vehicle control group value, respectively, on DSs 106 to 134.
Relative to body weight, male rats in the 150 mg/kg/day dosage group consumed significantly more (p≤0.05 or p≤0.01) feed between DS 50 and DS 84 and between DS 106 and DS 134, in comparison to the vehicle control group values. In addition,
relative feed consumption values were significantly increased (p≤0.05 or p≤0.01) in the 75 mg/kg/day dosage group on DSs 78 to 84 and DSs 127 to 134, in comparison to the vehicle control group value. These increases reflected the reductions in body weight and body weight gain that occurred in the 150 mg/kg/day dosage group during these same periods.

FEMALES:
Precohabitation
Absolute and relative feed consumption values in the 150 mg/kg/day dosage group were significantly reduced (p≤0.05 or p≤0.01) during the first week of the premating dosage period (DSs 1 to 8), in comparison to the vehicle control group values (8% and 6% less than the vehicle controls, respectively). These reductions correlated with the statistically significant reductions (p≤0.01) in body weight gain that occurred at 150 mg/kg/day during the same period.
Absolute and relative feed consumption values during the premating period were unaffected by the 25 and 75 mg/kg/day dosages of cyclamen aldehyde. Absolute feed consumption values in the 25, 75 and 150 mg/kg/day dosage groups were 98%, 96% and 95% of the vehicle control group value, respectively, for the premating dosage period (DSs 1 to 15). Relative feed consumption values in the 25, 75 and 150 mg/kg/day dosage groups were 99%, 98% and 97% of the vehicle control group value, respectively, during the same period.

Gestation
Absolute and relative feed consumption values during the gestation period were unaffected by dosages of cyclamen aldehyde as high as 150 mg/kg/day. All values were comparable among the four dosage groups and did not significantly differ. Absolute feed consumption values in the 25, 75 and 150 mg/kg/day dosage groups were 101%, 99% and 97% of the vehicle control group value, respectively, for the entire gestation period (DGs 0 to 21). Relative feed consumption values in the 25, 75 and 150 mg/kg/day dosage groups were 102%, 99% and 103% of the vehicle control group value, respectively, during the same period.

Lactation
Absolute and relative feed consumption values were significantly reduced (p≤0.05 or p≤0.01) in the 75 and 150 mg/kg/day dosage groups during the lactation period (DSs 1 to 15), in comparison to the vehicle control group values. Within the lactation period, absolute and relative feed consumption values were reduced (often statistically significant at p≤0.01) in the 75 and 150 mg/kg/day dosage groups at each tabulated interval within the lactation period. Absolute feed consumption values in the 25, 75 and 150 mg/kg/day dosage groups were 95%, 89% and 80% of the vehicle control group value, respectively, on DLs 1 to 15. Relative feed consumption values in the 25, 75 and 150 mg/kg/day dosage groups were 96%, 91% and 84% of the vehicle control group value, respectively,during the same period.
Absolute and relative feed consumption values during the lactation period were unaffected by the 25 mg/kg/day dosage of cyclamen aldehyde.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Cyclamen aldehdye increased the absolute and relative weights of the epididymides (left, right and cauda) at 150 mg/kg/day. The increased epididymal weights generally reflected the presence of masses on the cauda epididymis, as previously described, and microscopic observations of moderate to marked sperm granulomas. Adrenal cortical atrophy, affecting the zona fasciculate and zona reticularis.

There were microscopic test substance-related pathology findings observed in the male rats evaluated. In male rats given 150 mg/kg/day of cyclamen aldehyde the gross epididymal masses correlated microscopically with moderate to marked sperm
granulomas, associated with mild to moderate epithelial degeneration. The testes were not affected. In addition, these male rats had decreased adrenal weights that correlated microscopically with minimal adrenal cortical atrophy, affecting the zona fasciculate and zona reticularis. These adrenal and epididymal gross or microscopic changes were not seen in the male rats in the lower dosage groups (25 and 75 mg/kg/day). Additional changes seen in these rats were considered to be incidental or spontaneous changes commonly observed in control Crl:CD(SD) rats.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The number of estrous stages per 14 days was comparable among the four dosage groups before the start of administration and during the precohabitation period.
All mating and fertility parameters [numbers of days in cohabitation, rats that mated, the fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates during the first or second week of cohabitation and number of pregnancies per number of rats in cohabitation] were unaffected by dosages of cyclamen aldehyde as high as 150 mg/kg/day. All values were comparable among the four dosage groups and did not significantly differ.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Based on the individual data, motility of the sperm from the vas deferens could not be observed in 13 of 25 rats in the 75 mg/kg/day dosage group and any of the rats in the 150 mg/kg/day dosage group. The motility values in each of the samples generally reflected the presence of drifting debris, headless sperm, detached heads and/or less than the required number of sperm for evaluation. The effects observed at 150 mg/kg/day correlated with the infertility that was observed in the treated male rats that were mated with the untreated cohort female rats that were previously described. Only 12 rats in the 75 mg/kg/day dosage group had enough viable sperm (at least 200 sperm in 20 fields) available for analysis. The sperm motility values from the 12 treated male rats in the 75 mg/kg/day were comparable to the vehicle control group values and were also within the ranges observed historically at the Testing Facility.
.
The 75 mg/kg/day dosage of cyclamen aldehyde significantly reduced (p≤ 0.05) the sperm count and density from the cauda epididymis, as compared to the vehicle treated group values. Each of these average values were within the within the ranges observed historically at the Testing Facility.

All sperm parameters evaluated were unaffected by the 25 mg/kg/day dosage of cyclamen aldehyde.

Reproductive performance:

no effects observed

Description (incidence and severity):

FEMALES:
Pregnancy occurred in 25 (100.0%), 24 (96.0%), 24 (96.0%) and 24 (96.0%) of the 25 mated female rats in the 0 (Vehicle), 25, 75 and 150 mg/kg/day dosage groups, respectively. All pregnant dams delivered litters. At 150 mg/kg/day, the average number of implantation sites per delivered litter was significantly reduced (p≤0.01), in comparison to the vehicle control group value (13.8 implantation sites vs. 15.5 implantation sites in vehicle controls). However, the average value was within the range observed historically at the Testing Facility .
The number of dams with all pups dying between days 1 and 5 postpartum was significantly increased (p≤0.01) in the 150 mg/kg/day dosage group, in comparison to the vehicle control group. Reflecting the reduction in implantation sites, the average number of pups delivered per litter as well as the average number of liveborn pups per litter was significant reduced (p≤0.01) in the 150 mg/kg/day dosage group, in comparison to the vehicle control group values. In addition, there was a significant increase (p≤0.01) in the number of stillborn pups that were delivered in the 150 mg/kg/day dosage group (8 stillborn pups vs. 3 stillborn pups in the vehicle controls). However, the average values for the total number of delivered pups, the number of liveborn pups and the number of stillborn pups were within the range observed historically at the Testing Facilitya. Pup mortality (i.e., found dead, presumed cannibalized or unscheduled sacrifice) was significantly increased (p≤0.01) in the 150 mg/kg/day dosage group on days 1 to 5 postpartum, in comparison to the vehicle control group values. As a result of the increase in pup mortality, the overall viability index (i.e., number of live pups on day 5 postpartum/number of liveborn pups on day 1 postpartum) was significantly reduced (p≤0.01) in the 150 mg/kg/day dosage group, in comparison to the vehicle control group value (75.7% vs. 96.3% in the vehicle controls). At 150 mg/kg/day, the averages for the number of surviving pups per litter and the live litter size at weighing was significantly reduced (p≤0.01) on days 1 through 22 postpartum, in comparison to the vehicle control group values. In addition, the average pup body weight per litter was significantly reduced (p≤0.05 or p≤0.01) in the 75 and 150 mg/kg/day dosage groups at each tabulated interval between days 1 and 22 postpartum, as compared to the vehicle control group values.

No other natural delivery and litter observations were affected by dosages of cyclamen aldehyde as high as 150 mg/kg/day. Values for the numbers of dams delivering litters, the duration of gestation, the gestation index (number of dams with one or more liveborn pups/number of pregnant rats), the numbers of dams with stillborn pups, lactation index, and percent male pups per number of pups sexed per litter were comparable among the four dosage groups.
The statistically significant increase (p≤0.01) in pup mortality observed on days 12 through 15 postpartum and the corresponding reduction (significant at p 0.01) in the lactation index that occurred at 25 mg/kg/day was considered unrelated to cyclamen aldehyde because the changes were not dose related.

Ovarian Follicle Counts
Although the typical effect of a test substance upon ovarian follicle production and growth is depletive, the noted effect of an increase in the number of follicles may also be relevant. Significant decreases in individual (left and right) ovarian weights were observed at 150 mg/kg/day, as previously described. The increased number of primordial follicles may inversely correlate to these decreased organ weights in Group IV (150 mg/kg/day) rats because the smaller ovaries may cause a more concentrated dispersion of follicles than in the larger ovaries of the Control (Group I) rats. However, since no microscopic correlates were reported for the histopathology of these rat ovaries, biological relevance of the increased number of follicles could not be completely defined. Corpora lutea were present in all rats evaluated.

In treated male rats, reproductive organ weights were reduced at both 75 and 150mg/kg bw/day. Adverse effects on sperm analyses and histopathological changes to the epididymides were observed at 150mg/kg bw/day dose level. In treated female rats, reduced gestational body weights were observed at 150mg/kg bw/day dose level. Reduced pup body weights were also observed at both 150mg/kg bw/day dose levels. In untreated females mated to the treated males, a reduced number of implantation sites and a reduced fertility index were observed at th 150mg/kg bw/day dose level.

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

reproductive function (sperm measures)

Remarks on result:

other: n/a

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

reproductive performance

Remarks on result:

other: n/a

Critical effects observed:

yes

Lowest effective dose / conc.:

75 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

seminal vesicle

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 Generation Pups of Treated Male Rats Mated with Untreated Female Rats:
There were no litters produced at 150 mg/kg/day from the mating of treated P generation male rats with untreated female rats. Therefore, these parameters were not evaluated at 150 mg/kg/day. None of the clinical signs that occurred in the F1 generation pups were attributed to treatment of P generation male rats with cyclamen aldehyde at dosages as high as 75 mg/kg/day. All transient and persistent clinical observations were considered unrelated to cyclamen aldehyde because: 1) the incidences were not dosage-dependent; and/or 2) the number of litters affected did not differ significantly from the vehicle control group values.

F1 Generation Pups of Treated Female Rats Mated with Untreated Male Rats:
At 150 mg/kg/day, 20 of 24 litters (significant at p≤0.01) had one or more pups with a lenticular opacity in one or both eyes. This observation only occurred in one pup from one litter in the vehicle control group on days 19 through 22 postpartum. In the 150 mg/kg/day dosage group, lenticular opacities were first observed on day 16 and generally persisted until day 22 postpartum. In addition, this clinical sign was confirmed during scheduled necropsy examination.
There were no other treatment-related clinical signs observed in the F1 generation pups following treatment of P generation female rats with cyclamen aldehyde at dosages as high as 150 mg/kg/day. All transient and persistent clinical observations were considered unrelated to cyclamen aldehyde because: 1) the incidences were not dosage-dependent; and/or 2) the number of litters affected did not differ significantly from the vehicle control group values.

Postweaning:
- F1 Generation Rats of Treated Male Rats Mated with Untreated Female Rats:
As previously described, there were no litters produced at 150 mg/kg/day from the mating of treated P generation male rats with untreated female rats. Therefore, these parameters were not evaluated at 150 mg/kg/day. None of the clinical signs that occurred in the F1 generation male or female rats were attributed to treatment of P generation male rats with cyclamen aldehyde at any dosage level tested. Clinical signs that were observed in the F1 generation rats included mild dehydration (based on skin turgor), chromorhinorrhea, scabbing (neck, back, right forelimb or tail), an ulceration on the right axilla or back, chromodacryorrhea,urine-stained abdominal fur, slight excess salivation, bent tail, constrictions on the tail, and discoloration (purple) and an abrasion on the tail.
The significant increase (p≤0.01) in the number of F1 generation female rats in the 25 mg/kg/day paternal dosage group with a scab was considered unrelated to paternal treatment with cyclamen aldehyde because the increase was not dose related.

- F1 Generation Rats of Treated Female Rats Mated with Untreated Male Rats:
At 150 mg/kg/day, the number of F1 generation male and female rats with a lenticular opacity in one or both eyes was significantly increased (p≤0.01), in comparison to the vehicle control group value. This observation was first observed in this dosage group during the preweaning period, as previously described. In addition, this observation was more prevalent in F1 generation male rats than in the female rats (18 male rats vs. 6 female rats).

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1 Generation Rats of Treated Male Rats Mated with Untreated Female Rats:
As previously described, there were no litters produced at 150 mg/kg/day from the mating of treated P generation male rats with untreated female rats. Therefore, these parameters were not evaluated at 150 mg/kg/day. Body weight gains in the F1 generation male and female rats were unaffected by paternal treatment with cyclamen aldehyde at 25 and 75 mg/kg/day. Body weight gains in the F1 generation male rats were 105% and 104% of the vehicle control group value at 25 and 75 mg/kg/day, respectively, on days 23 to 57 postpartum. In F1 generation female rats, body weight gains were 101% and 103% of the vehicle control group value at 25 and 75 mg/kg/day, respectively, during the same period.

The average body weight in the F1 generation male rats on day 57 postpartum was 105% and 104% in the 25 and 75 mg/kg/day paternal dosage group, respect vely. In the F1 generation female rats, the average body weight on day 57 postpartum was 102% in the 25 and 75 mg/kg/day paternal dosage groups.

F1 Generation Rats of Treated Female Rats Mated with Untreated Male Rats:
In F1 generation male rats, body weight gains were significantly reduced (p≤0.01) in the 150 mg/kg/day maternal dosage group on days 23 to 30 postpartum and days 30 to 37 postpartum, in comparison to the vehicle control group values. Thereafter, body weight gains were comparable to the vehicle control group values during the remainder of the postweaning period. Reflecting the initial reductions in weight gain, body weight gains in the F1 generation male rats in the 150 mg/kg/day maternal dosage group were 94% (significant at p≤0.05) of the vehicle control group value for the entire postweaning period (days 23 to 57 postpartum). In F1 generation female rats, body weight gains were significantly reduced (p≤0.05) in the 150 mg/kg/day maternal dosage group on days 23 to 30 postpartum, in comparison to the vehicle control group value. However, this reduction was transient, and body weight gains were comparable to the vehicle control group values during the remainder of the postweaning period. Body weight gains in the 25, 75 and 150 mg/kg/day maternal dosage group were 101%, 98% and 100% of the vehicle control group value, respectively, on days 23 to 57 postpartum. Reflecting significant reductions (p≤0.05 or p≤0.01) in the average pup body weight per litter that occurred prior to weaning, the average body weight were also significantly reduced (p≤0.05 or p≤0.01) in the F1 generation male and female rats on days 23, 30, 37, 44, 51 and/or 57 postpartum in the 75 and 150 mg/kg/day dosage groups. All other statistically significant reductions were considered unrelated to maternal treatment with cyclamen aldehyde because the reductions were transient and had no overall effect on the cumulative body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- F1 Generation Rats of Treated Male Rats Mated with Untreated Female Rats:
There were no litters produced at 150 mg/kg/day from the mating of treated P generation male rats with untreated female rats. Therefore, these parameters were not evaluated at 150 mg/kg/day.Absolute and relative feed consumption values in the F1 generation male and female rats were unaffected by paternal treatment with cyclamen aldehyde at 25 and 75 mg/kg/day. Absolute feed consumption values in the F1 generation male rats were 107% and 103% of the vehicle control group value at 25 and 75 mg/kg/day, respectively, on days 23 to 57 postpartum. In F1 generation female rats, absolute feed consumption values were 101% of the vehicle control group value at 25 and 75 mg/kg/day during the same period.

- F1 Generation Rats of Treated Female Rats Mated with Untreated Male Rats:
Corresponding to significant reductions in body weight gains, absolute feed consumption values in the F1 generation male rats were significantly reduced (p≤0.05 or p≤0.01) in the 150 mg/kg/day maternal dosage group on days 23 to 30 postpartum and days 30 to 37postpartum, in comparison to the vehicle control group values. Relative to body weight, F1 generation male rats consumed significantly more (p≤0.05 or p≤0.01) feed on days 30 to 37 and 37 to 44 postpartum at 75 and 150 mg/kg/day and days 44 to 51, 51 to 57 and 23 to 57 postpartum at 150 mg/kg/day.

Similar observations occurred in the F1 generation female rats, in that, absolute feed consumption values were significantly reduced (p≤0.01) in the 150 mg/kg/day maternal dosage group on days 23 to 30 postpartum, in comparison to the vehicle control group value. Relative to body weight, F1 generation female rats consumed significantly more (p≤0.05 or p≤0.01) feed on days 37 to 44 and 23 to 57 postpartum at 75 and 150 mg/kg/day and days 44 to 51 and 51 to 57 postpartum at 150 mg/kg/day.

Absolute feed consumption values in the F1 generation male rats were 98%, 97% and 93% of the vehicle control group value at 25, 75 and 150 mg/kg/day, respectively, on days 23 to 57 postpartum. In F1 generation female rats, absolute feed consumption values were 98%, 99% and 96% of the vehicle control group value at 25, 75 and 150 mg/kg/day, respectively, during the same period.

Sexual maturation:

no effects observed

Description (incidence and severity):

- F1 Generation Rats of Treated Male Rats Mated with Untreated Female Rats:
As previously described, there were no litters produced at 150 mg/kg/day from the mating of treated P generation male rats with untreated female rats. Therefore, these parameters were not evaluated at 150 mg/kg/day. There were no effects on sexual maturation (preputial separation or vaginal opening) at any paternal dosage level tested. The average day on which sexual maturation was achieved and the average body weight on the day criterion was met was comparable among the dosage group and within the ranges observed historically at the Testing Facility.

- F1 Generation Rats of Treated Female Rats Mated with Untreated Male Rats:
There were no effects on sexual maturation (preputial separation or vaginal opening) at any maternal dosage level tested. The average day on which sexual maturation was achieved was comparable among the dosage group and within the ranges observed historically at the Testing Facilitya. The average body weight of male rats on the day preputial separation occurred was significantly reduced (p≤0.05 or p≤0.01) in the 75 and 150 mg/kg/day dosage groups, in comparison to the vehicle control group value. These reductions in body weight reflect significant reductions (p≤0.05 or p≤0.01) in the average pup body weight per litter that occurred prior to weaning. Each of the average values for body weight on the day of
sexual maturation was within the ranges observed historically at the Testing Facilitya. Therefore, this finding was not considered an adverse effect of cyclamen aldehyde.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

- F1 Generation Pups of Treated Male Rats Mated with Untreated Female Rats:
Anogenital distance on days 1 or 22 postpartum in F1 male and female pups was not affected by treatment of P generation male rats with cyclamen aldehyde at any dosage level tested.
Nipple eruption did not occur in any male pup, and all female pups had nipples present on day 12 postpartum. There were no litters produced at 150 mg/kg/day from the mating of treated P generation male rats with untreated female rats. Therefore, these parameters were not evaluated at 150 mg/kg/day.


- F1 Generation Pups of Treated Female Rats Mated with Untreated Male Rats:
In male pups, anogenital distance on day 1 postpartum was not affected by treatment of P generation female rats with cyclamen aldehyde at any dosage level tested. On day 22 postpartum, there was a significant reduction (p≤0.05) in the anogenital distance of male pups in the 75 and 150 mg/kg/day dosage groups, in comparison to the vehicle control group value. When covaried with fetal body weights per litter, the statistically significant reduction (p≤0.05) in anogenital distance was not apparent in the 75 and 150 mg/kg/day dosage groups, in comparison to the vehicle control group value. This developmental delay correlated with an overall reduction in pup body weights on day 22 postpartum, as previously described. In female pups, anogenital distance on day 1 postpartum was not initially affected by treatment of P generation female rats with cyclamen aldehyde at any dosage level tested. However, when covaried with fetal body weights per litter, there was a statistically significant increase (p≤0.01) in anogenital distance at 150 mg/kg/day, in comparison to the vehicle control group value. This increase in anogenital distance was no longer apparent by day 22 postpartum. Nipple eruption did not occur in any male pup, and all female pups had nipples present on day 12 postpartum.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

F1 Generation Pups of Treated Female Rats Mated with Untreated Male
Rats
In male pups, anogenital distance on day 1 postpartum was not affected by treatment of
P generation female rats with cyclamen aldehyde at any dosage level tested. On
day 22 postpartum, there was a significant reduction (p≤0.05) in the anogenital distance
of male pups in the 75 and 150 mg/kg/day dosage groups, in comparison to the vehicle
control group value. When covaried with fetal body weights per litter, the statistically
significant reduction (p≤0.05) in anogenital distance was not apparent in the 75 and
150 mg/kg/day dosage groups, in comparison to the vehicle control group value. This
developmental delay correlated with an overall reduction in pup body weights
on day 22 postpartum, as previously described.
In female pups, anogenital distance on day 1 postpartum was not initially affected by
treatment of P generation female rats with cyclamen aldehyde at any dosage level tested.
However, when covaried with fetal body weights per litter, there was a statistically
significant increase (p≤0.01) in anogenital distance at 150 mg/kg/day, in comparison to
the vehicle control group value. This increase in anogenital distance was no longer
apparent by day 22 postpartum.
Nipple eruption did not occur in any male pup, and all female pups had nipples present
on day 12 postpartum.

Description (incidence and severity):

- F1 Generation Rats of Treated Male Rats Mated with Untreated Female Rats:
Tthere were no litters produced at 150 mg/kg/day from the mating of treated P generation male rats with untreated female rats. Therefore, these parameters were not evaluated at 150 mg/kg/day.
Terminal body weights in the F1 generation male and female rats were comparable among the three remaining dosage groups and did not significantly differ. There were no test substance-related changes in the absolute or relative (% terminal body
weight) weight of the reproductive organs or the pituitary, brain or adrenal glands of the F1 generation male or female rats at any dosage level tested.

- F1 Generation Rats of Treated Female Rats Mated with Untreated Male Rats:
There were no test substance-related changes in the absolute or relative (% terminal body weight) weight of the reproductive organs or the pituitary, brain or adrenal glands of the F1 generation male or female rats at any dosage level tested.

Gross pathological findings:

no effects observed

Description (incidence and severity):

F1 Generation Pups of Treated Male Rats Mated with Untreated Female Rats:
As previously described, there were no litters produced at 150 mg/kg/day from the mating of treated P generation male rats with untreated female rats. Therefore, no data is available at 150 mg/kg/day.
There were no gross lesions observed in the F1 generation pups that were stillborn or found dead or in the F1 generation pups that survived to scheduled necropsy on day 22 postpartum.

F1 Generation Pups of Treated Female Rats Mated with Untreated Male Rats:
The only gross lesion observed in the F1 generation pups was a tan area on the left kidney of one male pup (19694-3) in the 150 mg/kg/day. There were no other gross lesions observed in the F1 generation pups that survived to scheduled necropsy on day 22 postpartum. This gross lesion was not attributed to cyclamen aldehyde because it was limited to a single pup in the high dosage group. In the pups that were stillborn, found dead or humanely euthanized, no milk was present in the stomach of 1, 5, 5 and 14 F1 generation pups in the 0 (Vehicle), 25, 75 and 150 mg/kg/day dosage groups, respectively. The remaining pups that were stillborn, found dead or humanely euthanized appeared normal.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

Treated males mated to untreated females

Generation:

F1

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no aparent effect up to 75 mg/kg/d

Remarks on result:

other: n/a

Dose descriptor:

NOAEL

Remarks:

Treated females mated to untreated males

Generation:

F1

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: n/a

Reproductive effects observed:

not specified

150mg/kg body weight changes , Developmental effects , eye effects , liver , organ weight changes , reproductive effects

- P generation male treated rats results:

No treatment-related deaths were observed. One male rat at this dose level was sacrificed on DS 53 as a result of a broken palate. In addition, three male rats in the vehicle control group were found dead on DS 86, 123, 130. None of these deaths were attributed to treatment with the test material because the incidence was not dosage-dependent. All other P generation male rats survived to scheduled sacrifice. Clinical signs attributed to the test material were limited to slight or moderate excess salivation at this dose level. This observation occurred intermittently during the dosage period and was not considered an adverse effect of the test material. Reductions in bodyweight gain occurred intermittently during the dosage period prior to cohabitation, with statistically significant reductions occurring on DSs 50 to 57 and DSs 64 to 71. Body weight gains were also reduced at this dose level on DSs 84 to 92 at each tabulated interval between DS 99 and DS 134, in comparison to the vehicle control group values. Overall, body weight gains at this dose level were significantly reduced on DSs 1 to 134 and DS 1 to termination. The average body weight was slightly reduced beginning on DS 72 and continuing until scheduled sacrifice, reaching statistical significance on DS 120 when compared to the vehicle control group value. There were no apparent effects of the test material on feed consumption in the P generation male rats at this dose level. The test material caused infertility following mating with untreated female rats at this dose level. There was only one pregnancy (1/24) produced by matings at this dose level. Terminal body weights for P generation male rats at this dose level were slightly reduced, in comparison to the vehicle control group value. This reduction did not reach statistical significance, but reflected the statistically significant reduction in body weight gains that occurred in this dosage group for the cumulative dosage period. The test material increased the absolute and relative weights of the epididymides (left, right and cauda) at this dose level. The increased epidiymal weights generally reflected the presence of masses on the cauda epidiymis and microscopic observations of moderate to marked sperm granulomas. In addition, the absolute and relative weights of the liver were increased or significantly increased at this dose level, as compared to the vehicle control group values. The absolute weight of the adrenal glands was significantly reduced at this dose level in comparison to the vehicle control group value. The decreased adrenal weights correlated microscopically with minimal adrenal cortical atrophy, affecting the zona fasciculate and zona reticualris. Sperm motility from sperm taken from the vas deferens could not be observed in all rats at this dose level. The motility values in each of the samples generally reflected the presence of drifting debris, headless sperm, detached heads and/or less than the required number of sperm for evaluation. The effects correlated with the infertility that was observed in the treated male rats that were mated with the untreated cohort female rats. Pregnancy occurred in 23 and 1 of the 25 to 28 untreated female rats that were assigned to mate with treated male rats in the 0 (vehicle) and 150 mg/kg/day dose level, respectively. The pregnant dam at this dose level did not deliver a litter.

- P generation female rat results:

all rats survived to scheduled sacrifice. No clinical signs related to the test material were observed in the treated P generation female rats during the premating, gestation and lactation period. During the first week of the dosage period (DSs 1 to 8), body weight gains were significantly reduced as compared to the vehicle controls. Despite the rebound during the second week of the dosage period, body weight gains remained significantly reduced for the entire premating dosage period (DSs 1 to 15) as compared to the vehicle control group value. In gestating rats at this dose level, body weight gains were reduced at each tabulated interval within the gestation dosage period. Although the reductions within the gestation period did not reach statistical significance, the cululative material body weight gains (DGs 0 to 21) were significantly reduced as compared to the vehicle control group value. The average maternal body weight on DG 18 and DG 21 was significantly reduced at this dose level compared to the vehicle control group value. At the beginning o the lactation period (DLs 1 to 5), body weight losses were observed in each test material treated group compared to gains in the vehicle control group during the same period. The losses in body weight were statistically significant at this dose level. Weight gains remained significantly reduced during the next tabulated interval (DLs 5 to 8) compared to the vehicle control group value. The average maternal body weight on DLs 5 to 11 was significantly reduced at this dose level in comparison with the vehicle control group values. During the premating period, absolute and relative feed consumption values at this dose level were slightly, but significantly reduced on DSs 1 to 8 in comparison to the vehicle control group values. These reductions correlated with the statistically significant reductions in body weight gain at this dose level during the same period. These reductions in feed consumption were transient and did not persist during the gestation period. However, during the lactation period, absolute and relative feed consumption values were reduced (often statistically significant) at this dose level at each tabulated interval within the lactation period and overall for DLs 1 to 15 in comparison to the vehicle control group values. There were no apparent effects of the test material on estrous cyclicity or mating and fertility parameters at this dose level. Pregnancy occurred in 24 or 25 of the 25 mated female rats in the 0 (vehicle) and 150 mg/kg/day dose level, respectively. All pregnant dams delivered litters. The test material significantly reduced the average number of implantation sites per delivered litter at this dose level, in comparison to the vehicle control group value. However, the average value for implantation sites per delivered litter was within the range observed historically at the testing facility. Other effects on natural delivery at this dose level included: a significant increase in the number of dams with all pups dying between days 1 and 5 postpartum; a significant reduction in the average number of pups delivered per litter and the average number of liveborn pups per litter; a significant increase in the number of stillborn pups that were delivered; a significant increase in pup mortality on days 1 to 5 postpartum with a corresponding significant reduction in the viability index; and a significant reduction in the averages for the number of surviving pups per litter and the live litter size at weighing at on days 1 through 22 postpartum. The average values for the total number of delivered pups, the number of liveborn pups and the number of stillborn pups were within the range observed historically at the testing facility. In addition, the average pup body weight per litter was significantly reduced at this dose level between days 1 and 22 postpartum as compared to the vehicle control group values. There were no test material-related necropsy observations. Terminal body weights were comparable among the dosage groups and did not significantly differ. The test material significantly increased the absolute and relative weights of the liver in dams at this dose level. The absolute and relative weights of the non-gravid uterus (with the cervix) was significantly decreased and the weights of the left and right ovary were significantly decreased at this dose level as compared to the vehicle control group values. There were no microscopic changes in the uterus or ovaries that could be correlated with the differences in these organ weights. At this dose level, an increase in the number of primordial follicles was present in comparison to the corresponding control group. The increased primordial follicles may be inversely correlated to the decreased ovarian weights at this dose level because the smaller ovaries may cause a more concentrated dispersion of follicles than in the larger ovaries of the rats in the control group. However, since no microscopic correlated were reported for the histopathology of these rat ovaries, biological relevance of the increased number of follicles could not be completely defined. Corpora lutea were present in all rats evaluated. F1 generation pus of treated male rats mated with untreated female rats results: there were no litters produced at this dose level from mating of treated P generation male rats with untreated female rats. F1 generation pups of treated female rats mated with untreated male rats: a significant number of litters (20/24 litters) had one or more pups with a lenticular opacity in one or both eyes. The lenticular opacities were first observed on day 16 and generally persisted until day 22 postpartum and was confirmed during necropsy examination of pups not selected for continued evaluation. During the postweaning period, the number of F1 generation male and female rats with a lenticular opacity in one or both eyes was significantly increased at this dose level in comparison to the vehicle control group value. Ths observation was more prevalent in F1 generation male rats than in the female rats. IN male pups, anogenital distance on day 1 postpartum was not affected by treatment of P generation female rats with the test material. On day 22 postpartum, there was a significant reduction in the anogenital distance of male pups at this dose level in comparison to the vehicle control group.

 

25 mg/kg liver , no observed adverse effect level , organ weight changes dose was mg/kg/day. P generation treated male rats results at 25 mg/kg/day:

there were no treatment-related deaths. One male rat at this dose level was sacrificed on day 65 as a result of a broken palate. In addition, three male rats in the vehicle control group and one male at this dose level were found dead on DS 86, 105, 123, or 130. None of these deaths were attributed to treatment with the test material because the incidence was not dosage-dependent. All other P generation male rats survived to scheduled sacrifice. No clinical signs attributed to the test material were observed at this dose level. There were no apparent effects of the test material on body weight, feed consumption, organ weights, sperm motility, sperm count and density from the cauda epididymis at this dose level in P generation treated male rats. Pregnancy occurred in 23 and 24 of 25 to 28 untreated female rats that were assigned to mate with treated male rats in the 0 (vehicle) and 25 mg/kg/day dosage groups respectively. All pregnant dams in the 0 (vehicle) and 25 mg/kg/day dosage groups delivered litters. Natural delivery and litter observations were unaffected by dosages of the test material at this dose level. P generation female rats results at 25 mg/kg/day: all P generation treated female rats survived to scheduled sacrifice. No clinical signs related to the test material were observed during the premating, gestation and lactation periods. At the beginning of the lactation period (DLs 1 to 5) bodyweight losses were observed in the test material treated group compared to gains in the vehicle control group during the same period. Body weight gains rebounded in the test material treated group during the next tabulated interval (DLs 5 to 8). No effects were observed on food consumption, estrous cyclicity, mating or fertility parameters. Pregnancy occurred in 24 or 25 of the 25 mated female rats in the 0 (vehicle) and 25 mg/kg/day dosage groups, respectively (mated with untreated male rats). All pregnant dams delivered litters. Natural delivery and litter observations were unaffected by dosages of the test material at this dose level. There were no test material related necropsy observations. Terminal body weights were comparable among the dosage groups and did not signficantly differ. The test material significaintly increased the absolute and relative weights of the liver in this effect. Results of F1 generation pups of treated male rats mated with untreated female rats: none of the clinical signs that occurred in F1 generation pups during the preweaning or postweaning periods were attributed to treatment of P generation male rats with test material at this dose level. Anogenital distance, nipple eruption and sexual maturation were not affected by paternal treatment with the test material and there were no gross lesions in the F1 generation pups or rats that were attributed to the test material. Body weights (including terminal body weights), body weight gains and feed consumption values in the F1 generation male and female rats were unaffected by paternal treatment with the test material at this dose level. There were no test material related changes in the absolute or relative (% terminal weight) weight of the reproductive organs or the pituitary, brain or adrenal glands of the F1 generation male or female rats. Results from F1 generation pups of treated female rats mated with untreated male rats: in male pups, anogenital distance on day 1 postpartum was not affected by treatment of P generation female rats with test material at any dose level tested. In female pups, anogenital distance on day 1 postpartum was not affected by treatment of P generation female rats with the test material at this dose level. There were no gross lesions in the F1 generation pups or rats that were attributed to the test material at this dose level. There were no effects on F1 generation male or female rat body weights, body weight gains or feed consumption at this dose level when compared to vehicle control values. There were no effects on sexual maturation (preputial separation or vaginal opening) at any maternal dose level tested. Terminal bodyweights of F1 generation male and female rats were comparable at this dose level when compared to the vehicle control group. There were no test material substance-related changes in the absolute or relative (% terminal body weight) weight of the reproductive organs or the pituitary, brain or adrenal glands in either sex at any dosage tested. The reproductive NOAEL in the P generation male and female rats is 25 mg/kg/day. The NOAEL for general toxicity in P generation female rats is 25 mg/kg/day. The NOAEL for viability and growth of the F1 generation offspring of treated P generation female rats is 25 mg/kg/day.

75 mg/kg body weight changes , liver , no observed adverse effect level , organ weight Changes

dose was mg/kg/day. P generation treated male rats results at 55 mg/kg/day: there were no treatment-related deaths. Three male rats in the vehicle control group were found dead on DS 86, 123, and 130. None of these deaths were attributed to treatment with the test material. All other P generation male rats survived to scheduled sacrifice. No clinical signs attributed to the test material were observed at this dose level. There were no apparent effects of the test material on body weight, feed consumption at this dose level in P generation treated male rats. The absolute and relative weights of the liver were increased or significantly increased at this dose level as compared to the vehicle control group values. The absolute weight of the adrenal glands was significantly reduced at this dose level when compared to the vehicle control group. The decreased adrenal weights correlated microscopically with minimal adrenal cortical atrophy, affecting the zona fasciculate and zona reticularis. Sperm motility from sperm taken from the as deferens could not be observed in 13/25 rats at this dose level. The motility values in each of the samples generally reflected the presence of drifting debris, headless sperm, detached heads and/or less than the required number of sperm for evaluation. The test material significantly reduced the sperm count and density from the cauda epididymis as comapred to the vehicle treated group values. The average values for sperm historically at the testing facility. Pregnancy occurred in 23 of 25 to 28 untreated female rats that were assigned to mate with treated male rats in the 0 (vehicle) and 75 mg/kg/day dosage groups. All pregnant dams in the 0 (vehicle) and 75 mg/kg/day dosage groups delivered litters. Natural delivery and litter observations were unaffected by dosages of the test material at this dose level. P generation female rats results at 25 mg/kg/day: all P generation treated female rats survived to scheduled sacrifice. No clinical signs related to the test material were observed during the premating, gestation and lactation periods. During the first week of the dosage period (DSs 1 to 8) body weight gains were reduced at this dose level as compared to vehicle controls. Despite the rebound during the second week of the dosage period, body weight gains at this dose level remained reduced for the entire premating dosage period (DSs 1 to 15) as compared to the vehicle control group value. At the beginning of the lactation period (DLs 1 to 5) bodyweight losses were observed in the test material treated group compared to gains in the vehicle control group during the same period. Body weight gains rebounded in the test material treated group during the next tabulated interval (DLs 5 to 8). During the lactation period, absolute and relative feed consumption values were reduced (often statistically significant) at this dose level at each tabulated interval within the lactation period and overall for DLs 1 to 15 in comparison to the vehicle control group values. There were no apparent effects of the test material on estrous cyclicity, mating or fertility parameters at this dose level. Pregnancy occurred in 24 or 25 of the 25 mated female rats in the 0 (vehicle) and 75 mg/kg/day dosage groups, respectively (mated with untreated male rats). All pregnant dams delivered litters. Natural delivery were unaffected by dosages of the test material at this dose level. The average pup body weight per litter was significantly reduced at this dsoe level at each tabulated interval between days 1 and 22 postpartum as compared with the vehicle control values. There were no test material related necropsy observations. Terminal body weights were comparable among the dosage groups and did not signficantly differ. The test material significaintly increased the absolute and relative weights of the liver in dams treated at this dose level. There were no microscopic changes to correlate this effect. Results of F1 generation pups of treated male rats mated with untreated female rats: none of the clinical signs that occurred in F1 generation pups during the preweaning or postweaning periods were attributed to treatment of P generation male rats with test material at this dose level. Anogenital distance, nipple eruption and sexual maturation were not affected by paternal treatment with the test material and there were no gross lesions in the F1 generation pups or rats that were attributed to the test material. Body weights (including terminal body weights), body weight gains and feed consumption values in the F1 generation male and female rats were unaffected by paternal treatment with the test material at this dose level. There were no test material related changes in the absolute or relative (% terminal weight) weight of the reproductive organs or the pituitary, brain or adrenal glands of the F1 generation male or female rats. Results from F1 generation pups of treated female rats mated with untreated male rats: in male pups, anogenital distance on day 1 postpartum was not affected by treatment of P generation female rats with the test material at any dosage level tested. On day 22 postpartum, there was a significant reduction in the anogenital distance of male pups at this dose level in comparison to the vehicle control group value. Whe covaried with fetal body weights per litter, the statistically sifniciant reduction in anogenital distance was no longer apparent. This developmental delay correlated with an overall reduction in pup weights on day 22 postpartum. In female pups, anogenital distance on day 1 postpartum was not initially affected by treatment of P generation female rats with the test material at this dose level. There were no gross lesions in the F1 generation pups or rats that were attributed to the test material. Reflecting significant reductions in the average pup body weight per litter that occurred prior to weaning, the average body weight were also significantly reduced in the F1 generation male and female rats on days 23, 30, 37, 44, 51 and/or 57 postpartum at this dose level. There were no effects on sexual maturation (preputial separation or vaginal opening) at any maternal dose level tested. In the F1 generation male and female rats, terminal bodyweights were comparable among the dosage groups and did not significantly differ. There were no test material related changes in the absolute or relative (% terminal body weight) weight of the reproductive organsor the pituitary, brain or adrenal glands in either sex at any dose level tested. The NOAEL for general toxicity in treated males mated to untreated female is 75 mg/kg/day. The NOAEL for F1 viability and growth in treated males mated to untreated females is 75 mg/kg/day. body weight changes , Developmental effects , eye effects , liver , organ weight changes , reproductive effects dose was 150 mg/kg/day (cond't). F1 generation pups of treated female rats mated with untreated male rats: When covaried with fetal body weights per litter, the statistically significant reduction in anogenital distance was no longer apparent. This developmental delay correlated with an overall reduction in pup body weights on day 22 postpartum. In female pups, anogenital distance on day 1 postpartum was not initially affected by treatment of P generation female rats with the test material. However, when covaried with fetal body weights per litter, there was a statistically significant increase in anogenital distance at this dose level in comparison to the control group value. This increase in anogenital distance was no longer apparent by day 22 postpartum. There were no gross lesions in the F1 generation pups or rats that were attributed to the test material. In F1 generation male rats, body weight gains were significantly reduced at this maternal dose group on days 23 to 30 postpartum, days 30 to 37 postpartum and overall for the entire postweaning period (days 23 to 57 postpartum) in comparison to the vehicle control group values. Transient, but statistically significantly reductions in body weight gains occurred in the F1 generation female rats on day 23 to 30 postpartum in comparison to the vehicle control group value. Reflecting significant reductions in the average pup body weight per litter that occurred prior to weaning, the average body weight were also significantly reduced in the F1 generation male and female rats on days 23, 30, 37, 44, 51 and/or 57 postpartum at this dose level. Corresponding to significant reductions in body weight gains, absolute feed consumption values were significantly reduced in the F1 generation male rats on days 23 to 30 postpartum and days 30 to 37 postpartum and in F1 generation female rats on days 23 to 30 postpartum at this dose level in comparison to vehicle control values. Relative to body weight, F1 generation male and female rats consumed significantly more feed overall for the entire postweaning period (days 23 to 57 postpartum) at this dose level. There were no effects on sexual maturation (preputial separation or vaginal opening) at any maternal dosage tested. Terminal body weights in the F1 generation male rats were significantly reduced at this dose level in comparison to vehicle control group value. In the F1 generation female rats, terminal bodyweights were comaprable among the dosage groups and did not significantly differ. There were no test material related changes in the absolute or relative (% terminal body weight) weight of the reproductive organs or the pituitary, brain or adrenal glands in either sex at any dosage level tested.

Conclusions:

The No-observable-adverse-effect-level (NOAEL) for general toxicity of the test material in P generation male rats is 75 mg/kg/day.
The reproductive NOAEL in the P generation male rats is 25 mg/kg/day.
The NOAEL for general toxicity of the test material in the P generation female rats is 25 mg/kg/day.
The reproductive NOAEL in the P generation female rats is 25 mg/kg/day.
The NOAEL for viability and growth of the F1 generation offspring of treated P generation male rats is 75 mg/kg/day.
The NOAEL for viability and growth of the F1 generation offspring of treated P generation female rats is 25 mg/kg/day.

Executive summary:

The purpose of this study was to provide information concerning the effects of cyclamen aldehyde on reproductive function of treated male and female rats, and on the growth and development of F1 generation offspring to day 60 postpartum.

Cyclamen aldehyde increased the incidence of excess salivation in P generation male rats given 150 mg/kg/day. However, this clinical sign was not considered an adverse effect of cyclamen aldehyde. At 150 mg/kg/day, reductions in body weights and body weight gains occurred in P generation male rats intermittently during the dosage period, in the absence of reductions in feed consumption. Gross lesions attributed to treatment with cyclamen aldehyde were observed in the reproductive organs (cauda epididymides, prostate and seminal vesicle) in P generation male rats given 150 mg/kg/day. Non-reproductive organ weights (i.e., liver and adrenal glands) at ≥75 mg/kg/day and terminal body weights at 150 mg/kg/day were affected in P generation male rats. The toxicological significance of the increased liver weights was unable to be determined because there were no microscopic findings that could be correlated with the changes in organ weights. However, the decreased adrenal weights correlated microscopically with minimal adrenal cortical atrophy, affecting the zona fasciculate and zona reticularis. Based on these data, the no-observable-adverse-effect-level (NOAEL) for general toxicity of cyclamen aldehyde in P generation male rats is 75 mg/kg/day.

The reproductive NOAEL in the P generation male rats is 25 mg/kg/day. Infertility, as determined following matings with untreated female rats, was observed at 150 mg/kg/day. In addition, increased epididymal weights, with corresponding microscopic observations of moderate to marked sperm granulomas, occurred at 150 mg/kg/day. Sperm motility was affected at ≥75 mg/kg/day, as well as epididymal sperm count and density at 75 mg/kg/day. There were no apparent effects on natural delivery parameters in the untreated female rats mated with treated male rats at 25 or 75 mg/kg/day.

In P generation female rats, cyclamen aldehyde affected body weights and/or body weight gains at ≥75 mg/kg/day during the premating period and at 150 mg/kg/day during the gestation and lactation periods. Corresponding reductions in feed consumption occurred only during the premating (150 mg/kg/day) and lactation periods (75 and 150 mg/kg/day). Non-reproductive organ weights (i.e., liver) were affected in P generation rats in each dosage group; however, the toxicological significance of the increased liver weights was unable to be determined because there were no microscopic findings that could be correlated with the changes in organ weights. Based on these data, the no-observable-adverse-effect-level (NOAEL) for general toxicity of cyclamen aldehyde in P generation female rats is 25 mg/kg/day.

The reproductive NOAEL in the P generation female rats is also 25 mg/kg/day. Cyclamen aldehyde did not affect estrous cyclicity or mating and fertility parameters at any dosage level. However, P generation female rats given 150 mg/kg/day cyclamen aldehyde had fewer implantation sites and delivered pups, increased pup mortality, as well as reduced pup survival and litter size. At ≥75 mg/kg/day, the average pup body weight was reduced throughout the preweaning period. Cyclamen aldehyde reduced the weight of the uterus at ≥75 mg/kg/day and ovaries at 150 mg/kg/day; however, no microscopic correlates were noted in either organ. The average number of primordial follicles was increased in P generation female rats at 150 mg/kg/day; however, the biological relevance of this finding could not be determined. The NOAEL for viability and growth of the F1 generation offspring of treated P generation male rats is 75 mg/kg/day. There were no apparent effects on body weights, feed consumption, anogenital distance, nipple eruption, sexual maturation or organ weights (reproductive and non-reproductive) up to 75 mg/kg/day.

The NOAEL for viability and growth of the F1 generation offspring of treated P generation female rats is 25 mg/kg/day. In utero and lactation exposure to cyclamen aldehyde at 150 mg/kg/day caused an increase of lenticular opacities during the preweaning period that persisted into the postweaning period. By day 22 postpartum, anogenital distance was reduced in male pups at 75 and 150 mg/kg/day, but this delay in development may have reflected the overall reduction in pup body weights prior to weaning and is not considered to be a direct effect of cyclamen aldehyde. In female pups, anogenital distance was increased at 150 mg/kg/day based on covaried analysis; however, this increase was no longer apparent by day 22 postpartum. Body weights, body weight gains and feed consumption values were affected in both sexes at ≥75 mg/kg/day during the postweaning period. There were no apparent effects on nipple eruption, sexual maturation or organ weights (reproductive and non-reproductive) up to 150 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study has been performed to recognised standards (GLP, OECD 415) however the results were determined to be due to a metabolite of this parent molecule, and not of the molecule itself by further studies described below.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The effects previously seen with Cyclamen aldehyde in rats were thought not to be relevant to human metabolism and exposure.

An in vitro metabolism study in hepatocytes, comparing rat to human, rabbit and mouse, showed clear differences in metabolism between the rat and other species. The study was conducted at 3 doses (1, 10 and 100μm) and 3 timepoints (0, 1 and 4 hours treatment). The toxic metabolite thought to be responsible for the reproductive organ effects seen in rats (4-isopropyl benzoic acid) is found as a metabolite in rat hepatocytes but is not found in human, rabbit or mouse.

The overall data indicate that the rabbit would provide a better model for human toxicity of Cyclamen aldehyde. Confirmation of the presence of the toxic metabolite (4-isopropyl benzoic acid) in rats in vivo had also been previously detected in rat urine during a gavage study.

Effects on developmental toxicity

Description of key information

OECD 415: P generation female rats given 150 mg/kg/day cyclamen aldehyde had fewer implantation sites and delivered pups, increased pup mortality, as well as reduced pup survival and litter size in the presence of maternal toxicity.

At ≥75 mg/kg/day, the average pup body weight was reduced throughout the preweaning period. Cyclamen aldehyde reduced the weight of the uterus at ≥75 mg/kg/day and ovaries at 150 mg/kg/day; however, no microscopic correlates were noted in either organ. The average number of primordial follicles was increased in P generation female rats at 150 mg/kg/day; however, the biological relevance of this finding could not be determined.

OECD 414: In maternal animals, no adverse effects due to cyclamen aldehyde were observed but a slight decrease in maternal body weight during gestation was reported at the high dose.

In the fetuses, the lower mean fetal body weight (up to 9.5%) observed in the 150 mg/kg bw/d dose group was considered as an adverse effect, and led to a NOAEL for developmental toxicity of 75 mg/kg bw/d. These adverse effects on fetuses were observed in the presence of a slight decrease in maternal body weight during gestation.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Following ECHA decision (CCH-D-2114361700-57-01/F) on3-p-cumenyl-2-methylpropionaldehyde, EC No 203-161-7, it was requested to conduct additional toxicological studies:
1. In vitro cytogenicity study in mammalian cells (Annex VIII, Section 8.4.2., test method: OECD TG 473) or in vitro micronucleus study (Annex VIII, Section 8.4.2, test method: OECD TG 487
2. In vitro gene mutation study in mammalian cells (Annex VIII, Section 8.4.3.; test method: OECD TG 476 or TG 490) with the registered substance provided that the study requested under 1. has negative results;
3. Sub-chronic toxicity study (90-day), oral route (Annex IX, Section 8.6.2.; test method: EU B.26./OECD TG 408) in rats with the registered substance;
4. Pre-natal developmental toxicity study (Annex IX, Section 8.7.2.; test method: EU B.31./OECD TG 414) in a first species (rat or rabbit), oral route with the registered substance;
5. Extended one-generation reproductive toxicity study (Annex IX, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance after providing the OECD 408 outcome.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

Exceptions to GLPs: characterization of the test substance performed by the Sponsor according to established SOPs, controls, and approved test methodologies to ensure integrity and validity of the results generated

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han

Details on test animals or test system and environmental conditions:

Test animals: time-mated female Crl:WI(Han) rats were received from the lab on Gestation Day 1, 2, 3, or 4. The animals were approximately 12–13 weeks old and weighed between 186 and 240 g at the initiation of dosing on Gestation Day 6.

Housing: single/individual housing in solid-bottom cages containing appropriate bedding material. Cages were arranged on the racks in group order. Where
possible, control group animals were housed on a separate rack from the test substance-treated animals.

Food:
Type: Meal
Frequency: Ad libitum.
Analysis: Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Testing Facility. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water:
Type: Municipal tap water, treated by reverse osmosis and ultraviolet irradiation.
Frequency: Ad libitum, via an automatic watering system. Water bottles were provided, if required.
Analysis: Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

Acclimation period: After receipt at the Testing Facility, the Crl:WI(Han) rats were acclimated prior to the initiation of dosing.

Environmental conditions:
Temperature: 68°F to 77°F (20°C to 25°C)
Humidity: 30% to 70%
Light Cycle: 12 hours light and 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Dose formulations were divided into aliquots where required to allow them to be dispensed on each dosing occasion.
Vehicle was used as received and test substance was prepared daily. For both, the storage conditions were set to maintain 18°C to 24°C, protected from light, purged with nitrogen.
Dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The prepared formulations were not adjusted for purity.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis.

Concentration analyses:
Concentrations: all groups
Stratum: middle
Number of samples per concentration: 4 collected samples, 2 analyzed samples, 2 backup samples
Intervals: first and last preparation
Storage conditions: temperature set to maintain 18°C to 24°C.
Acceptance criteria: mean sample concentration within 100% ± 10% of theoretical concentration. Individual sample concentration of ± 15%.

Analyses were performed by a high performance liquid chromatography method with ultraviolet absorbance detection using a validated analytical procedure.

Details on mating procedure:

Time-mated rats were received by the lab on Gestation Day 1, 2, 3 and 4. The day on which confirmation of mating is made will be designated Gestation Day 0.

Duration of treatment / exposure:

Gestation Days 6–20

Frequency of treatment:

Once daily

Duration of test:

Gestation Days 0-21

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

75 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

No. of animals per sex per dose:

22 females/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

The oral route of exposure was selected because this a possible route of human exposure. The dose levels were selected based on information provided by the Sponsor. In a previous
1 generation reproduction toxicity study, gestating rats given 150 mg/kg/day of cyclamen aldehyde were observed with reduced mean body weight and body weight gains compared to the control group generally throughout gestation. Based on these results, 150 mg/kg/day was selected as the high dose for the current study. It was expected that this high dose would induce maternal toxicity while not resulting in death or severe suffering. Lower doses were selected to assess dose response.

Maternal examinations:

Mortality: checked at least twice daily (morning and afternoon), beginning upon arrival through termination. Animals were observed within their cage unless necessary for identification or confirmation of possible findings.

Clinical observations: checked once daily, beginning with the day of animal arrival and continuing through (and including) the day of euthanasia. Animals were removed from the cage. Mortality and all signs of overt toxicity were recorded on the day observed. The observations included, but were not limited to, evaluations for changes in appearance of skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous systems, somatomotor activity, and behavior. On days of scheduled dosing, the observations were collected prior to dosing.

Postdose observations (with animal handling): checked approximately 1 hour postdose. Animals were removed from the cage. Animals were observed for findings that were potentially related to treatment or that might change before the next scheduled observation.

Individual Body Weights: checked at Gestation Days 0 (by supplier) and 5–21 (daily). Gestation Day 0 bodyweight collected under Non-GLP conditions. Not collected from animals found dead.

Food Consumption: checked at Gestation Days 5–21 (daily). Quantitatively measured. Reported as g/animal/day for each corresponding body weight interval
during gestation.

Examinations after necropsy: liver and thyroid gland (organ weights, histology, histopathology)

Ovaries and uterine content:

The uterus of each dam was excised, and its adnexa trimmed. Corpora lutea were also counted and recorded. Gravid uterine weights were obtained and recorded. The uterus of each dam was opened, and the number of viable and nonviable fetuses, early and late resorptions, and total number of implantation sites were recorded, and the placentae were examined. The individual uterine distribution was documented using the following procedure: all implantation sites, including
early and late resorptions, were numbered in consecutive fashion beginning with the left distal uterine horn, noting the position of the cervix and continuing from the proximal to the distal right uterine horn. Uteri which appear nongravid by macroscopic examination were opened and placed in a 10% ammonium sulfide solution for detection of early implantation loss. Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible.

Blood sampling:

Thyroid Hormone Analyses:
Time: Gestation Day 21.
Special requirements: Collected prior to noon on each day of collection, around the same time each day, and within a 2-hour window on each collection day, to reduce variability due to normal diurnal variations in physiological levels of thyroid hormones. Blood collections were performed in an animal ante room, the necropsy laboratory, or as far away from live animals as possible to minimize stress-induced hormone fluctuations.
Method: Venipuncture from a jugular vein using the hand-held restraint method.
Target volume: approximately 1.0 mL/time point collected.
Anticoagulant: none
Processing: serum
Number of aliquots: 2
Analyses: for total T3 and T4 analyses, hormone samples were analyzed using validated ultra-high performance liquid chromatography with dual mass spectroscopy (UHPLC/MS/MS) assays. Analysis of serum samples to determine TSH concentrations was conducted using a validated Luminex Bead Based (TSH) assay.

Fetal examinations:

Fetal examinations were conducted without knowledge of treatment group. External, internal, and skeletal fetal findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal), malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life), or incidental (minor changes in coloration, mechanical damage to specimen, etc.).

External: Each viable fetus was examined in detail, sexed, weighed, tagged, and euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region. Following euthanasia, anogenital distance was measured for all viable fetuses. The absolute and relative values (to the cube root of fetal body weight) were reported. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible), and the tissue was discarded.

Internal (visceral): The sex of all fetuses was confirmed by internal examination. Approximately one-half of the fetuses in each litter were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The heads from these fetuses were removed and placed in Harrison’s fixative for subsequent processing and soft-tissue examination.

Skeletal: The remaining fetuses (approximately one-half from each litter, excluding any carcasses without heads) were eviscerated and fixed in 100% ethyl alcohol. Following fixation in alcohol, fetuses were stained with Alizarin Red S and Alcian Blue. The skeletal examination was made following this procedure.

Statistics:

Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Indices:

The following parental indices and litter calculations were included, where applicable:
Pre-Implantation Loss = (No. of corpora lutea – no. of implants) x 100/No. of corpora lutea
Post-Implantation Loss = (No. of implants – no. of live fetuses) x 100/No. of implants
Sex Ratio (% males) = No. male fetuses x 100/Total no. of fetuses
Litter % of Fetuses with Abnormalities = No. of fetuses in litter with a given finding x 100/No. of fetuses in litter examined

Historical control data:

Historical control data were available from the testing laboratory.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related clinical observations noted at any dose level at the daily examinations or approximately 1 hour postdose.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All females in the control, 25, 75, and 150 mg/kg/day groups survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 150 mg/kg/day group, a slight mean body weight loss with correspondingly lower mean food consumption were noted following the initiation of dosing (Gestation Days 6–7) compared to the control group. Mean body weight gains and food consumption were generally comparable to the control group for the remainder of the study and when the entire treatment period (Gestation Days 6–21) was evaluated and the mean absolute body weights in this group were unaffected. Therefore, the initial decrements in the 150 mg/kg/day group were considered test substance-related, but nonadverse.
Mean body weights and body weight gains in the 25 and 75 mg/kg/day groups and mean corrected body weights, corrected body weight gains in the 25, 75, and 150 mg/kg/day groups were unaffected by test substance administration.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 150 mg/kg/day group, a slight mean body weight loss with correspondingly lower mean food consumption were noted following the initiation of dosing (Gestation Days 6–7) compared to the control group. Mean body weight gains and food consumption were generally comparable to the control group for the remainder of the study and when the entire treatment period (Gestation Days 6–21) was evaluated and the mean absolute body weights in this group were unaffected. Therefore, the initial decrements in the 150 mg/kg/day group were considered test substance-related, but nonadverse.
Food consumption in the 25 and 75 mg/kg/day groups was unaffected by test substance administration.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Lower mean total T3 and T4 levels were observed in the 75 (-34.0% and -23.4% respectively) and 150 (-44.3% and -45.3% respectively) mg/kg/day groups compared to the control group; differences were statistically significant. Mean total T3 and T4 levels in the 25 mg/kg/day group were comparable to the control group mean.
Mean TSH levels in the 25, 75 and 150 mg/kg/day groups were higher than the control group mean; differences were non-dose responsive and statistically significant only for the 25 and 75 mg/kg/day groups. In addition, TSH mean values across all treated groups were within the range of the lab historical control data for Sprague Dawley rats and the control group mean was atypically low, and outside the range of the historical control data; when evaluated on an individual animal basis, more than 50% of the animals in the control group had TSH levels that fell outside of the range of the historical control data.
There were no differences in thyroid gland weights or any corresponding changes in thyroid histopathology in any of the treated groups when compared to the control group. However, test article-related higher mean (absolute) liver weights were observed in the 75 and 150 mg/kg/day group females.
Based on these data, the lower T3 and T4 levels noted in the 75 and 150 mg/kg/day groups were likely attributable to increased hormone metabolism in the liver, rather than a direct effect on the thyroid gland, and hence considered nonadverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test article-related higher mean (absolute) liver weights were observed in the 75 and 150 mg/kg/day group females. Higher mean liver weights were not dose-responsive, but multiple individual absolute liver values in the 75 and 150 mg/kg/day group females were out of the concurrent control group range. There were no microscopic correlates.
No test substance-related organ weight changes were noted in the thyroid gland.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related gross pathology findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of Cyclamen Aldehyde.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, microscopic liver changes were present in the 25, 75, and 150 mg/kg/day group females and consisted of minimal hepatocellular single-cell necrosis. Single-cell necrosis was perivascular and/or random, and was characterized as individual eosinophilic rounded hepatocytes with pyknotic nuclei. Single-cell necrosis was dose-responsive in incidence rate.
Of note, a marginal increased incidence of focal or multifocal hepatocellular necrosis (n=4), characterized as random groups (vs. single-cell/individual) of necrotic hepatocytes with associated inflammatory infiltrates, was also observed in the 150 mg/kg/day group females, but was considered incidental given the observation of this change in the concurrent control group females (n=2), and frequent occurrence of this change in both the lab historical control database and in untreated rats used in toxicity studies (Thoolen et al., 2010; McInnes, 2011).
There were no test substance-related microscopic changes in the thyroid gland. Remaining changes were considered incidental, of the nature commonly observed in this strain and age of
rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of Cyclamen Aldehyde.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

There were no adverse effects on maternal parameters observed at any dosage level.

Number of abortions:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dose levels of 25, 75 and 150 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss,
mean number of live fetuses, and fetal sex ratios.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Intrauterine survival was unaffected by test substance administration at dose levels of 25, 75 and 150 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss,
mean number of live fetuses, and fetal sex ratios.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dose levels of 25, 75 and 150 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss,
mean number of live fetuses, and fetal sex ratios.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dose levels of 25, 75 and 150 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss,
mean number of live fetuses, and fetal sex ratios.

Dead fetuses:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance administration at dose levels of 25, 75 and 150 mg/kg/day. Parameters evaluated included mean litter proportions of postimplantation loss,
mean number of live fetuses, and fetal sex ratios.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Details on maternal toxic effects:

There were no adverse effects on maternal parameters observed at any dosage level.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 150 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

dead fetuses

early or late resorptions

endocrine findings

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal body weights (males, females, and combined) in the 75 and 150 mg/kg/day groups were lower than the concurrent control group (3.978%–5.913% and 8.539%–9.499%, respectively) and below the mean values in the lab historical control data. The lower mean fetal body weights in these groups were considered test substance-related and adverse at 150 mg/kg/day based on the magnitude of change from the control group, but nonadverse at 75 mg/kg/day due to the low magnitude of change from the control group and lack of any fetal morphological effects.
Intrauterine growth at 25 mg/kg/day was unaffected by test substance administration.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Mean anogenital distances (absolute and relative to the cube root of the fetal weight) were unaffected by test substance administration at all dose levels. Differences from the control group were slight and not statistically significant.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No external developmental malformations were observed for fetuses in the test substance-treated groups. In the control group, Fetus No. 1505-01 was noted with a thread-like tail which consisted
skeletally of absent sacral and caudal vertebrae. No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related skeletal developmental malformations were noted. In the 150 mg/kg/day group, a malformation of bent humerus was observed for a single fetus (Fetus No. 4501-12); variations of bent scapula, wavy ribs, thoracolumbar supernumerary ribs, and incomplete ossification of the frontal, parietal, squamosal, and supraoccipital bones of the skull were also observed for this fetus. As the bent limb bone was only observed for a single fetus, it was not considered to be test substance-related.
No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the lab historical control data.

Visceral malformations:

no effects observed

Description (incidence and severity):

No visceral developmental malformations were observed in fetuses in this study.
No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the lab historical control data.
A discolored liver lobe was noted for Fetus No. 2503-04 in the 25 mg/kg/day group and was not classified as either a malformation or variation. This finding was considered incidental and was not considered to be test substance-related because it occurred infrequently and/or in a manner hat was not dose-related.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

In the 75 and 150 mg/kg/day groups, test substance-related lower (up to 5.913% and 9.499%, respectively) mean fetal body weights (males, females, and combined) were noted compared to the control group. Based on the magnitude of change versus the control group, the effects on mean fetal body weights were considered nonadverse at 75 mg/kg/day but adverse at 150 mg/kg/day. There were no test substance-related effects on intrauterine growth at 25 mg/kg/day or intrauterine survival and fetal morphology (external, visceral, and skeletal) at 25, 75, and 150 mg/kg/day.
The numbers of fetuses (litters) available for morphological evaluation were 177(20), 186(19), 202(20), and 209(22) in the control, 25, 75, and 150 mg/kg/day groups, respectively.
Malformations were observed in 1 fetus each in the control and 150 mg/kg/day groups and were considered spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance-treated groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the lab historical control data ranges. Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

In the 150 mg/kg/day group, a malformation of bent humerus was observed for a single fetus (Fetus No. 4501-12); variations of bent scapula, wavy ribs, thoracolumbar supernumerary ribs, and incomplete ossification of the frontal, parietal, squamosal, and supraoccipital bones of the skull were also observed for this fetus. As the bent limb bone was only observed for a single fetus, it was not considered to be test substance-related.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

no

Conclusions:

In conclusion, there were no adverse effects on maternal parameters observed at any dosage level but a slight decrease in maternal body weight during gestation was reported at the high dose. Therefore, a dose level of 150 mg/kg/day, the highest dose level evaluated, was considered to be the NOAEL for maternal toxicity. Based on lower mean fetal weights at 150 mg/kg/day, a dose level of 75 mg/kg/day was considered to be the NOAEL for developmental toxicity when cyclamen aldehyde was administered orally by gavage to time-mated Crl:WI(Han) rats. These low mean fetal weights occurred in the presence of a slight decrease in maternal body weight during gestation at the high dose.

Executive summary:

The objectives of this study were to determine the potential of Cyclamen Aldehyde to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a NOAEL (no-observed-adverse-effect level) for maternal and developmental toxicity.

Animals were dosed with Cyclamen Aldehyde at doses of 25, 75 and 150 mg/kg bw/d via oral gavage once daily during Gestation Days 6–20.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, thyroid hormones, gross necropsy findings, organ weights, intrauterine growth and survival, and fetal morphology.

In the maternal animals, test substance-related effects included a slight mean body weight loss with correspondingly lower mean food consumption in the 150 mg/kg/day group immediately following the initiation of dosing (Gestation Days 6–7). Mean body weight gains and food consumption in this group were generally comparable to the control group for the remainder of the study and the initial decrements did not impact the mean absolute body weights, and therefore were considered nonadverse. Test substance-related lower total T3 and T4 levels were observed in the 75 and 150 mg/kg/day groups. These differences were considered nonadverse based on lack of corresponding changes on thyroid gland weights or thyroid histopathology in these groups. Test substance-related higher mean liver weights were observed at 75 and 150 mg/kg/day with no microscopic correlate. Based on these data, the lower T3 and T4 levels observed at 75 and 150 mg/kg/day groups were likely attributable to increased hormone metabolism in the liver, rather than a direct effect on the thyroid gland, and hence considered nonadverse. Additionally, no test substance-related effect on TSH levels were noted at any dose level. Although higher TSH levels were noted in the 25, 75, and 150 mg/kg/day groups, the differences from the control group were non-dose responsive, statistically significant for only the 25 and 75 mg/kg/day groups, and within the range of the lab historical control data for Sprague Dawley rats. Additionally, minimal single-cell necrosis was observed dose-responsively in all test substance groups but considered nonadverse given the minimal severity grade and absence of an associated inflammatory response.

In the 75 and 150 mg/kg/day groups, test substance-related lower (up to 5.9% and 9.5%, respectively) mean fetal body weights (males, females, and combined) were noted compared to the control group. Based on the magnitude of change versus the control group, the effects on mean fetal body weights were considered nonadverse at 75 mg/kg/day but adverse at 150 mg/kg/day. There were no test substance-related effects on intrauterine growth at 25 mg/kg/day or intrauterine survival and fetal morphology (external, visceral, and skeletal) at 25, 75, and 150 mg/kg/day.

In conclusion, there were no adverse effects on maternal parameters observed at any dosage level but a slight decrease in maternal body weight during gestation was reported at the high dose. Therefore, a dose level of 150 mg/kg/day, the highest dose level evaluated, was considered to be the NOAEL for maternal toxicity. Based on lower mean fetal weights at 150 mg/kg/day, a dose level of 75 mg/kg/day was considered to be the NOAEL for developmental toxicity when cyclamen aldehyde was administered orally by gavage to time-mated Crl:WI(Han) rats. These low mean fetal weights occurred in the presence of a slight decrease in maternal body weight during gestation at the high dose.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study has been performed to recognised standards (GLP, OECD 414) however the results were determined to be due to a metabolite of this parent molecule, and not of the molecule itself by further studies described below.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

The effects previously seen with Cyclamen aldehyde in rats were thought not to be relevant to human metabolism and exposure.

An in vitro metabolism study in hepatocytes, comparing rat to human, rabbit and mouse, showed clear differences in metabolism between the rat and other species. The study was conducted at 3 doses (1, 10 and 100μm) and 3 timepoints (0, 1 and 4 hours treatment). The toxic metabolite thought to be responsible for the reproductive organ effects seen in rats (4-isopropyl benzoic acid) is found as a metabolite in rat hepatocytes but is not found in human, rabbit or mouse.

The overall data indicate that the rabbit (or mouse) would provide a better model for human toxicity of Cyclamen aldehyde. Confirmation of the presence of the toxic metabolite (4-isopropyl benzoic acid) in rats in vivo had also been previously detected in rat urine during a gavage study.

Mode of Action Analysis / Human Relevance Framework

Cyclamen aldehyde (CA) has been widely used for the last 100 years as a muguet note in perfumery. The safe use of this material is well established through the understanding of exposure and based on quantitative risk assessment confirmed by the RIFM Expert Panel, which is supported by a wide range of toxicology studies conducted over the last 20 years. Repeated dose studies in rats that were mainly conducted for the purposes of hazard identification for the REACH registration, revealed adverse effects on sperm maturation leading to impaired fertility as well as developmental effects (decrease in fetal body weight) in the presence of a slight decrease in maternal body weight during gestation. The spermatogenesis effects on rats appear to be linked to the main circulating metabolite, 4-isopropyl-benzoic acid (iPBA). However, metabolism studies in rat, rabbit and human primary cultures of suspended hepatocytes, indicated species differences with iPBA readily formed by rat hepatocytes but below detection limit in cells from rabbits and humans. In plated rat hepatocytes, iPBA is detected as Coenzyme A-conjugate and this conjugate (iPBA-CoA) accumulates to stable levels over 22 h. It has been shown, that in vitro accumulation of CoA-conjugates is a metabolic hallmark strongly correlated to male rat reproductive toxicity for a number of structurally related compounds. iPBA-CoA is also formed in vivo both in the liver and in the testes of rats dosed with CA. iPBA-CoA does not accumulate in plated rabbit and human hepatocytes where it is rapidly cleared within 22 h. In a rabbit in vivo study, no effects of CA on spermatogenesis were observed. Thus, a species specific metabolic fate linked to CA toxicity in male rats can be postulated based on analytical data in vitro and in vivo in the liver, and in male reproductive tissue in vivo. There is strong evidence that this species specific metabolic fate in the rat is not relevant to the rabbit, which is a non-responder species. Finally, lack of accumulation of iPBA-CoA in human hepatocytes indicates that like the rabbit, humans are unlikely to be vulnerable to iPBA hepatic and testicular toxicity.

Sources:

-Natsch et al., "A species specific metabolism leading to male rat reprotoxicity of cyclamen aldehyde: in vivo and in vitro evaluation", Food and Chemical Toxicology, 2021

-Laue et al., "Benzoyl‑CoA conjugate accumulation as an initiating event for male reprotoxic effects in the rat? Structure–activity analysis, species specificity, and in vivo relevance", Archives of Toxicology, 2020.

-Laue et al., "p-Alkyl-Benzoyl-CoA Conjugates as Relevant Metabolites of Aromatic Aldehydes With Rat Testicular Toxicity—Studies Leading to the Design of a Safer New Fragrance Chemical", Toxicological Sciences, 2017

Justification for classification or non-classification

Cyclamen Aldehyde (CA) has shown spermatogenesis effects in rats at 75 mg/kg/day from an OECD 415 study. However these effects are not observed in rabbits tested at 300 mg/kg/d during 28 days. CA has also shown, from an OECD 414 study, developmental effects on rat fetuses at 150 mg/kg/day, in the presence of a slight decrease in maternal body weight during gestation. Metabolism studies in rat, rabbit and human primary cultures of suspended hepatocytes, indicated species differences with 4 -isopropyl benzoic acid (iPBA) readily formed by rat hepatocytes but below detection limit in cells from rabbits and humans. It has been shown, that in vitro accumulation of CoA-conjugate is a metabolic hallmark strong correlated to male rat reproductive toxicity for a number of structurally related compounds. PBA-CoA is also formed in vivo both in the liver and in the testes of rats dosed with CA. iPBA-CoA does not accumulate in planted rabbit and human hepatocytes where it is rapidly cleared within 22 h. In a rabbit, in vivo study, no effects of CA on spermatogenesis were observed. Thus a species specific metabolic fate in the rat is not relevant to the rabbit, which is a non-responder species. Finally, lack of accumulation of iPBA-CoA in human hepatocytes indicates that like the rabbit, humans are unlikely to be vulnerable to iPBA hepatic and testicular toxicity. Therefore, the effects seen in rats are unlikely to be relevant to the human, and classification is not warranted.