Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive toxicity :

No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was considered to be 10mg/kg bw. When rats were treated with Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and -(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one (EC no 907 -706 -6) orally.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data from peer reviewed journal
Qualifier:
equivalent or similar to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
Reproductive toxicity study of the test chemical was performed on rats.
GLP compliance:
not specified
Limit test:
no
Justification for study design:
No data available
Specific details on test material used for the study:
- Name of test material: Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and -(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one
- Molecular formula: C13H20O
- Molecular weight: 192.3 g/mole
- Smiles : C1([C@@H](C(=CCC1)C)\C=C\C(C)=O)(C)C
- Inchl: 1S/C13H20O/c1-10-6-5-9-13(3,4)12(10)8-7-11(2)14/h6-8,12H,5,9H2,1-4H3/b8-7+
- Substance type: Organic
- Physical state: Liquid
Species:
rat
Strain:
not specified
Details on species / strain selection:
No data available
Sex:
female
Details on test animals and environmental conditions:
No data available
Route of administration:
oral: unspecified
Vehicle:
other: oil
Details on exposure:
Details on exposure
PREPARATION OF DOSING SOLUTIONS:
2 mg of test material in 0.1 ml oil solution was prepared


DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food )
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test material form a solution in oil
- Concentration in vehicle: 8-10mg/kg bw/day
- Amount of vehicle (if gavage): No data available

- Lot/batch no. (if required): No data available
- Purity: No data available
Details on mating procedure:
No data available
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
8 months
Frequency of treatment:
On alternate daily
Details on study schedule:
No data available
Remarks:
8-10 mg/kg bw
No. of animals per sex per dose:
No data available
Control animals:
not specified
Details on study design:
No data available
Positive control:
No data available
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data available
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: No data available
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

OTHER:
Oestrous cyclicity (parental animals):
No data available
Sperm parameters (parental animals):
No data available
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:No data available
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other:]Number of offspring, live pups, weight of pups at birth and after 7 and 21 days, and the viability of the pups after birth were observed.

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]No data available

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:No data available

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:No data available
Postmortem examinations (parental animals):
No data available
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.The F1 generation (offspring) were
allowed to reach maturity and Their offspring, the F2 generation were evaluated for reproduction parameters.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]Reproductive parameters were observed

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.No data available
Statistics:
No data available
Reproductive indices:
No data available
Offspring viability indices:
yes ,offspring viability were observed
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no effect was observed on body weight.
Food consumption and compound intake (if feeding study):
not specified
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:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
no effects was observed on number of pregnancies, number of offspring
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: No effect was observed in given dose
Critical effects observed:
no
System:
female reproductive system
Organ:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
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:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: No effects was observed at given dose
Critical effects observed:
no
System:
female reproductive system
Organ:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
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
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
no effects observed
Description (incidence and severity):
No effects were observed on Number of offspring, live pups, weight of pups at birth and after 7 and 21 days, and the viability of the pups after birth.
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
viability
body weight and weight gain
other: reproductive parameters
Remarks on result:
other: No effects was observed
Critical effects observed:
not specified
Reproductive effects observed:
no
Treatment related:
not specified
Conclusions:
No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was considered to be 10mg/kg bw. When rats were treated with the test chemical orally.
Executive summary:

The reproductive toxicity study of the test chemical was performed on rats . The test material was given 2 mg in 0.1 ml oil solution on alternate days for 8 months (equivalent to a dose of approximately 8–10 mg/kg body weight/day). The female rats were observed through three reproduction cycles for number of pregnancies, weight, number of offspring, live pups, weight of pups at birth and after 7 and 21 days, and the viability of the pups after birth. The F1 generation (offspring) were allowed to reach maturity and treated with 15 mg/kg of test material prior to being subject to reproductive toxicity testing. Their offspring; the F2 generation were evaluated for reproduction parameters. No adverse effect on any of the parameters measured. Hence No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was considered to be 10mg/kg bw. When rats were treated with the test chemical orally.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data is from a Klimisch 2 database and provides a robust study summary.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproductive toxicity

In different studies, Reaction mass the test chemical has been investigated for reproductive toxicity to a greater or lesser extent. Often are the studies based on in vivo experiments in rodents, i.e. most commonly in rats for the test chemical.

Reproductive toxicity

In different studies, Reaction mass the test chemical has been investigated for reproductive toxicity to a greater or lesser extent. Often are the studies based on in vivo experiments in rodents, i.e. most commonly in rats for the test chemical.

In experimental study given by D. Belsito et al.( Food and Chemical Toxicology 45 (2007) S130–S167) and J. Lalko, A. Lapczynski , D. McGinty, S. Bhatia, C.S. Letizia, A.M. Api (Food and Chemical Toxicology 45 (2007) S251–S257) The reproductive toxicity study of the test chemical was performed on rats . The test material was given 2 mg in 0.1 ml oil solution on alternate days for 8 months (equivalent to a dose of approximately 8–10 mg/kg body weight/day). The female rats were observed through three reproduction cycles for number of pregnancies, weight, number of offspring, live pups, weight of pups at birth and after 7 and 21 days, and the viability of the pups after birth. The F1 generation (offspring) were allowed to reach maturity and treated with 15 mg/kg of test material prior to being subject to reproductive toxicity testing. Their offspring; the F2 generation were evaluated for reproduction parameters. No adverse effect on any of the parameters measured. Hence No Observed Adverse Effect Level (NOAEL) for reproductive toxicity was considered to be 10mg/kg bw. When rats were treated with the test chemical orally.

It is further supported by experimental study conducted by Calvin C Willhite(Toxicology and Applied Pharmacology 83, 563–575.) on structurally similar test chemical. In a one-generation study, the teratogenic potency of the test chemical was evaluated in timed-pregnant LHK:LVG(SYR) hamsters. A single oral dose of the test chemical at 0, 48, 240 or 480 mg/kg/day in Tween 20: acetone was administered on Day 8 of pregnancy at the rate of 0.5 ml/kg body weight. The animals were sacrificed on day 14 of pregnancy. There were no clinical signs of toxicity due to the test chemical. The test chemical failed to induce signs of retinoid intoxication in the dams and the maternal weight gain was not affected significantly. There was no significant effect on incidence of abnormal litters, or mean litter fetal body weight or the other parameters observed when exposed to 48 or 240 mg/kg/day. At 480 mg/kg/day, one abnormal litter was observed. No significant effect on frequency of malformed fetuses, Malformation Hind limb lateral rotation and ankylosis were observed in fetuses of treated dams as compared to control. Therefore, NOAEL was considered to be 480 mg/kg bw for P and F1 generation when LHK: LVG female hamster were treated with the test chemical once orally by gavage.

 It is supported by experimental study conducted by B. L. Oser, S. Carson And Mona Oser (Fd Cosmet. Toxicol. Vol. 3, pp. 563-569, 1965) on structurally similar test chemical. In reproductive toxicity study of FDRL male and female rats treated with α- Ionone orally by feed. The test substance was diluted in cotton-seed oil in a concentration sufficient to provide the predetermined dosage in 2% of the diet. The oil solutions were incorporated into a nutritionally adequate basal ration (Purina Laboratory Chow). No effects were observed on body weight and body weight gain, food consumption and efficiency of food utilization of male and female rats as compared to control. Estimated compound intake for male was 11.8 mg/Kg bw/day and for female were 11.1 mg/Kg bw/day. Slightly increased in haematocrit level was observed in female rat as compared to control. The observed effect was considered to be non-significant. Similarly, no effect was observed on liver and kidney weight of treated rats as compared to control. In addition, No significant gross pathological and histopathological changes were observed on reproductive gonads in treated male and female rats as compared to control. Therefore, NOAEL was considered to be 11.8 mg/Kg bw/ day for male and 11.1 mg/Kg bw/ day for female when FDRL male and female rats treated with the test chemical orally by feed for 90 days.

In another study, the method in the publication was used to evaluate the reproductive and developmental toxicity of the test chemical. In this method, the test chemical was administered into the rats by oral gavage in the concentrations of 0. 250, 500, 750 and 1000 mg/kg bw/day. The vehicle used to gavage the test chemical was corn oil. The source for the animals was Center for breeding of laboratory animals of the Oswaldo Cruz Foundation (Cecal-Fiocruz) and weighed around 200 -300 g at the initiation of the study. The rats were kept in the controlled temperature environment wherein the humidity was maintained around 70% and photoperiod was adjusted to 12 hours dark and 12 hours light. The rats were housed in standard plastic cages with stainless steel cover lids and wood shavings as bedding and kept under conditions of controlled temperature. The animals were mated in the ratio of 2:1 (2 females and 1 males) and were cohabited for 2 hours. The pregnancy was confirmed by the presence of sperm in the vaginal smear and was referred to as day '0' of pregnancy. On day 21 of gestation all dams were anesthetized and killed by CO2 inhalation. The gravid uterus was weighed with its contents and the number of implantation sites, living and dead fetuses, and resorptions was recorded.The ratio of resorptions per implantations, and the percentage of resorptions per implantations per litter were substantially increased, while the ratio of live fetuses per implantations per litter was drastically decreased, in dams treated with the highest dose of the test chemical (1000 mg/kg bw/day). In maternal animals, no signs of toxicity was observed till 750 mg/kg bw/day. The animals gained normal weight until sacrifice. After sacrifice and gross necropsy, the gravid uterus weight did not differ significantly till 750 mg/kg bw/day. Although, at 1000 mg/kg bw/day dose group, the number of resorptions were observed to be increased in maternal animals. Also, the gravid uterine weight was observed to be decreased significantly as compared to the control group. Also, the ratio of implantation sites to resorptions was found to be lower as compared to the control group. Thus, based on all the findingsin the study, after administration of the test chemical it was observed that the NOAEL was found to be 750 mg/kg bw/day and LOAEL was found to be 1000 mg/kg bw/day.

Thus, based on the above mentioned studies for target substance the test chemical it was considered that no adverse effects on reproductive function were observed. Therefore, according to CLP criteria, the test chemical cannot be classified as reproductive and developmental toxicity at dose concentration 10mg/kg bw /day. So, based on the above mentioned studies for the test chemical and to its read across substance, it was considered that no adverse effects on sexual function and fertility was observed. Thus,comparing this value with the criteria of CLP regulation the test chemical can be "Not classified" for reproductive toxicity.

 

Effects on developmental toxicity

Description of key information

Developmental Toxicity Study:

No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 10mg/kg bw. When rats were treated with Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and -(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one (EC no 907 -706 -6) orally.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Reason / purpose:
read-across: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
A study of the test chemical was performed on rats to evaluate any adverse effects on developmental parameters.
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material: Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and -(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one
- Molecular formula: C13H20O
- Molecular weight: 192.3 g/mole
- Smiles : C1([C@@H](C(=CCC1)C)\C=C\C(C)=O)(C)C
- Inchl: 1S/C13H20O/c1-10-6-5-9-13(3,4)12(10)8-7-11(2)14/h6-8,12H,5,9H2,1-4H3/b8-7+
- Substance type: Organic
- Physical state: Liquid
Species:
rat
Strain:
not specified
Details on test animals and environmental conditions:
Study 1:
TEST ANIMALS
- Source: Oswaldo Cruz
Foundation Central Animal House breeding stock.
- Age at study initiation: 60 days old
- Weight at study initiation: Approx. 200 g
- Fasting period before study: No Data
- Housing: Rats were housed individually in standard plastic cages with stainless-steel coverlids and white pinewood shavings as bedding.
- Diet (e.g. ad libitum): A pelleted diet (Nuvital® for laboratory rats and mice was provided ad libitum.
- Water (e.g. ad libitum): Filtered tap water were provided ad libitum.
- Acclimation period: No Data Available
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 °C
- Humidity (%): Approx. 70%
- Air changes (per hr): No Data
- Photoperiod (hrs dark / hrs light): 12 hours dark; 12 hours light
IN-LIFE DATES: From: To: No Data

Study 2:
Details on test material -
- Name of test material (as cited in study report): Alpha-iso-methylionone
- Molecular formula (if other than submission substance): C14-H22-O
- Molecular weight (if other than submission substance): 206.327 g/mol
- Substance type: Organic
- Physical state: Liquid
- Impurities (identity and concentrations): No Data Available

Study 3:
CAS name: trans beta-ionone
CAS no: 79-77-6
IUPAC name: 4-[2,6,6-trimethyl-1-cyclohexen-1- yl]-3-buten-2-one
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Study 1:
Details on exposure
PREPARATION OF DOSING SOLUTIONS: The doses were prepared in the concentration of 0, 125, 250, 500, and 1000 mg/kg bw/day in corn oil (3.75 g/kg body wt/day).

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data
- Mixing appropriate amounts with (Type of food): NO Data
- Storage temperature of food: No Data

VEHICLE
- Justification for use and choice of vehicle (if other than water): No Data
- Concentration in vehicle: No Data
- Amount of vehicle (if gavage): 3.75 g/kg body wt/day).
- Lot/batch no. (if required): No Data
- Purity: No Data

Study 2:
Details on exposure
PREPARATION OF DOSING SOLUTIONS: Dosing formulations were prepared daily from bulk materials. Samples from each concentration of the dosing suspensions (first and last days of treatment) were analyzed for AIM content.

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data Available
- Mixing appropriate amounts with (Type of food): No Data Available
- Storage temperature of food: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water): No Data Available
- Concentration in vehicle: No Data Available
- Amount of vehicle (if gavage): No Data Available
- Lot/batch no. (if required): lot numbers 103K0107 and 074K0025; Sigma-Aldrich, St. Louis, MO
- Purity: No Data Available

Study 3:
Details on Exposure:
Duration of test: Approx.28 days
Frequency: Once; Single dose
Dose Concentration: 250, 500, 750 and 1000 mg/kg
body wt/day
No of animals: No Data
Details of control: Only Corn Oil was given to the animals in the control group.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No Data Available
Details on mating procedure:
Study 1:
Details of mating
- M/F ratio per cage: 2 female: 1 male
- Length of cohabitation: 2 hours
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: The presence of sperm in the vaginal smear. The day on which spermatozoa were found in the smear was designated as day 0 of pregnancy.
- After … days of unsuccessful pairing replacement of first male by another male with proven fertility: No Data
- Further matings after two unsuccessful attempts: [no / yes (explain)]: No Data
- After successful mating each pregnant female was caged (how): No Data
- Any other deviations from standard protocol: No Data

Study 2:
Details of mating
- M/F ratio per cage: 1 male : 1 female
- Length of cohabitation: 5 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Sperm was found in the vaginal smear or a copulatory plug was found in the vagina. The presence of spermatozoa and/or a copulatory plug in situ was designated as gestational day 0 (GD 0).
- After … days of unsuccessful pairing replacement of first male by another male with proven fertility: No Data Available
- Further matings after two unsuccessful attempts: [no / yes (explain)]: No Data Available
- After successful mating each pregnant female was caged (how): No Data Available
- Any other deviations from standard protocol: No Data Available

Study 3:
Details on mating:
- M/F ratio per cage: two females:one male
- Length of cohabitation: 2 hours
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Pregnancy confirmed by the presence of sperm in the vaginal smear.
Duration of treatment / exposure:
Study 1:
Duration of exposure:
Duration of test: Approx. 28 days
Frequency: Daily from GD 6 to 15.
Dose Comcentration: 0, 125, 250, 500, and 1000 mg/kg bw/day
No of animals: 0 mg/kg bw/day: 17 animals
125 mg/kg bw/day: 17 animals
250 mg/kg bw/day: 17 animals
500 mg/kg bw/day: 18 animals
1000 mg/kg bw/day: 20 animals

Study 2: The animals were exposed to the test chemical from gestation day 7 to gestation day 17.

Study 3: Animals were dosed once with a single dose.
Frequency of treatment:
Study 1: Daily from GD 6 to 15.
Study 2: Daily; Once
Study 3: Once; Single Dose
Duration of test:
Study 1: Approx. 28 days
Study 2: Approx. 27 days
Study 3: Approx. 28 Days
No. of animals per sex per dose:
Study 1:
0 mg/kg bw/day: 17 animals
125 mg/kg bw/day: 17 animals
250 mg/kg bw/day: 17 animals
500 mg/kg bw/day: 18 animals
1000 mg/kg bw/day: 20 animals

Study 2: 25 animals per group were used.

Study 3: No Data Available
Control animals:
yes, concurrent vehicle
Details on study design:
Study 1: No Data Available

Study 2:
Details on study design-
- Dose selection rationale: Doses were selected based on the 90-day subchronic study performed by RIFM.
- Rationale for animal assignment (if not random): The healthy, mated female rats, were assigned to four dosage groups, 25 rats/group, using a computer-generated (weight-ordered) randomization procedure based on body weights recorded.

Study 3: No Data Available
Maternal examinations:
Study 1: Maternal animals were observed for mortality and clinical signs.

Study 2:
-Animals were observed twice daily for viability and examined for abnormal clinical signs, abortions, and premature deliveries before dosage administration and approximately 1 h later.
-Body weights were recorded prior to the start of the study and daily during the dosage and postdosage periods.
-Feed consumption was recorded on GDs 0, 7, 10, 12, 15, 18, and 21.

Study 3: The rats were weighed on days 0, 11 and 21 of pregnancy. On day 21 of gestation all dams were anesthetized and killed by CO2 inhalation.
Ovaries and uterine content:
Study 1: The gravid uterus was quickly removed, and weighed with its contents. Ovaries were excised and cleared of adhering fat, and the number of corpora lutea graviditatis was counted. The uterine wall was carefully open and the number of living and dead fetuses and resorptions was counted. A method was employed to identify all sites of implantations including those that were completely resorbed. Briefly, the opened uterus was immersed in a 10% ammonium sulfate solution for 10 min, rinsed in running water and then immersed for additional 10 min in a solution containing equal parts of 2% potassium ferricyanide and 1% hydrochloric acid.

Study 2: Uteri of apparently nonpregnant rats were examined while pressed between glass plates to confirm the absence of implantation sites. Uteri from pregnant rats were excised and examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. The number of corpora lutea in each ovary was also recorded.

Study 3: The gravid uterus was weighed with its contents and the number of implantation sites, living and dead fetuses, and resorptions was recorded.
Fetal examinations:
Study 1: dead fetus was a nonlive fetus with discernible digits in any or all paws. All living fetuses were weighed, examined under a stereomicroscope for externally visible abnormalities, and numbered with a marker pen. One-third of the fetuses from each litter -chosen at random-was fixed in Bouin's solution for further evaluation of soft tissue anomalies using a sectioning technique. Heart, thymus, liver, spleen, kidneys and lungs of sectioned fetuses were also weighed. The remaining fetuses were fixed in a 5% formalin solution, macerated in potassium hydroxide, cleared with a glycerin–KOH solution and stained with Alizarin Red S for skeletal evaluation. Soft-tissue and skeleton abnormalities were recorded using an internationally harmonized terminology glossary for developmental anomalies in laboratory animals.

Study 2: Fetuses were removed from the uterus, weighed, and examined for gender and gross external alterations. Live fetuses then were euthanized by an intraperitoneal injection of pentobarbital before undergoing further examination. Approximately half of the fetuses in each litter were fixed in Bouin’s solution and examined for soft tissue alterations, using a variation of Wilson’s sectioning technique. (The remaining fetuses in each litter were eviscerated, cleared, stained with alizarin red S, and examined for skeletal alterations.

Study 3: The living fetuses were weighed, numbered with a marker pen, examined for externally visible anomalies under a stereomicroscope and fixed in a 5% formalin solution. The externallyvisible skull defects were scored for severity by using a grading scale as follows:
0 - absence of skull defects;
1 - very small opening on the skull;
2 - clearly defined opening on the skull with meningocele;
3 - slight protusion of the brain through a wider opening on the skull (mild exencephaly);
4 - pronounced protusion of the brain outside the skull opening (severe exencephaly).
Statistics:
1: Data were evaluated by one-way analysis of variance (ANOVA) and Bonferroni post hoc test or by Kruskal- Wallis test followed by the Mann-Whitney U test, whenever the assumptions of the one-way ANOVA were not fulfilled. When no more than two means were compared, the unpaired two-tailed Student t-test or the Mann-Whitney U test was used. Values are the mean ± standard deviation, or the median and range. Proportions were analyzed by the Chi-square test or by the Fisher exact test if any expected frequency was smaller than 5. In any case, a difference was considered to be significant when p≤0.05.
2: Data generated during the course of study were recorded using the Argus Automated Data Collection and Management System and the Vivarium Temperature and Relative Humidity Monitoring System. All data were tabulated, summarized, and/or statistically analyzed using the above systems in conjunction with Microsoft Excel (Microsoft Office 97, version SR-2) and/or The SAS System (version 6.12). Clinical observation and other proportion data were analyzed using the variance test for homogeneity of the binomial distribution.Continuous data were analyzed by using Bartlett’s test of homogeneity of variances and the analysis of variance. Dunnett’s test was used to identify statistical significance of individual groups. If the analysis of variance was not appropriate, the Kruskal-Wallis test or Dunn’s method of multiple comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s exact test was used to analyze the data.
3: The data were analysed by the one-way analysis of variance (ANOVA) followed by the Scheffe’s test or, alternatively, by the Kruskal/Wallis test followed by the Mann/Whitney U-test whenever the variable did not fit a normal distribution. Proportions were analysed by the x2-test or, alternatively, by the Fisher’s exact test.
Indices:
Study 1: Implantation Index, Fetal abnormality index, Fetal survival index, Resorption Index.
Study 2: Fetal Index, Implantation Index, Resorption Index.
Study 3: Implantation Index, Fetal Resorption Index, Fetal Survival Index.
Historical control data:
NO Data Available
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Study 1: Piloerection, vocalization and chromodacryorrhea were noted in rats treated with the highest dose of the test chemical (1000 mg/kg body wt/d). Only a few rats treated with the second highest dose of the test chemical (500 mg/kg body wt) presented red tears and one was hypoactive. No clinical signs was apparent after the period of treatment with the two highest doses and, at any time, among rats that received doses of the test chemical lower than 500 mg/kg body wt.

Study 2: Clinical signs that included occasional incidences of chromorhinorrhea, excess salivation, sparse hair coat, localized alopecia, urine-stained abdominal fur, soft or liquid feces, or decreased activity were observed in a few animals from each group of rats.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Description (incidence):
Study 1: All dams from control and test chemical treated groups survived to scheduled euthanasia on GD 21.

Study 2: No mortality occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Study 1: Except for a decrease in weight gain of dams of the highest dose group (1000 mg/kg bw/day) during the treatment period (GD 6–15), pregnancy weight gain of rats exposed to the test chemical did not differ from that of vehicle-treated controls.

Study 2: No effects were observed on body weights and body weight gain during the study period.
Food consumption and compound intake (if feeding study):
not specified
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
Description (incidence and severity):
Study 1: PostMortem Examinations did not reveal any abnormality in organ weights at any dose levels.

Study 2: There were no effects organs on ovary in maternal animals during study period.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Study 1: PostMortem Examinations did not reveal any abnormality or gross pathological changes at any dose levels.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Study 1: At 1000 mg/kg bw/day, the rats exhibited maximum adverse effects. Thus the NOAEL for maternal animals was considered to be 500 mg/Kg bw/day for maternal Wistar rats.

Study 2: The NOAEL for developmental toxicty of maternal animals was found to be 30 mg/kg bw/day.

Study 3: No overt signs of maternal toxicity and no dose-related reduction of pregnancy weight gain, after deduction of gravid uterus weight at term, were noted in dams treated with the test chemical.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Study 1: At 1000 mg/kg bw/day, the number of implantation sites per pregnant female was not altered. However, the percentage of rats that successfully copulated (treated females) and had implantation sites detectable on GD 21 (pregnant females) declined from nearly 94% in control animals to 60% among those treated with the highest dose of the test chemical (1000 mg/kg body wt/day) from GD 6 onwards. As, the implantation occurs on GD 6-8 might also indicate that the test chemical impaired the ongoing implantation and or induced very early post-implantation loss.

Study 3: The pre and post implantation loss was seen highest in 1000 mg/kg bw/day.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Study 1: The test chemical did not induce any litter losses and litter sizes at any given dose levels.

Study 3: At 1000 mg/kg bw/day, a total litter loss was observed by resorptions.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Study 1: The incidence of early, late and whole resorptions was found to be highest in the highest tested dose group (1000 mg/kg bw/day).

Study 3: Early or late resorptions were observed in 1000 mg/kg bw/day.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
Study 1: The number of dead fetuses was fpund to be increased in that of the highest tested dose group i.e. 1000 mg/kg bw/ day when compared to the control animals.

Study 3: Highest incidences of dead fetuses was observed in 1000 mg/kg bw/day dose group.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Other effects:
not specified
Details on maternal toxic effects:
Study 1: The maternal toxic effects were found to be highest in the 1000 mg/kg bw/day dose group.
Study 2: The NOAEL for developmental toxicty of maternal animals was found to be 30 mg/kg bw/day.
Study 3: The maximum incidences in the maternal development toxicity was found in 1000 mg/kg bw/day dose group.
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings
Remarks on result:
other: Not Specified
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings
Remarks on result:
other: Not Specified
Abnormalities:
not specified
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Study 3: The highest incidences of fetal body weight changes was observed in 1000 mg/kg bw/day.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Study 3: The highest incidence in decline of the fetal body weight changes was observed in 1000 mg/kg bw/day.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Study 1: The number of live offsprings were decreased at 1000 mg/kg bw/day.

Study 3: The reduction in the number of live offsprings was found to be the highest in 1000 mg/kg bw/day.
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
Study 1: At the highest dose group the sex ratio on GD21 slightly favoured male over female fetuses.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Study 3: The litter sizes and weights of pups in litter were significantly reduced in 1000 mg/kg bw/day.
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Study 2: In some sporadic cases, the only fetal gross external alterations occurred in one control fetus in which a depressed left eye bulge, a fleshy thoracic
protrusion, and the presence of an extra hind limb were observed

Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Study 1: No consistent dose-related increase in the incidence of fetal skeleton anomalies was noted in the offspring of the test chemical treated dams. Only in a few instances (e.g., Axis misshapen, right and left Spine scapula misshapen) was the occurrence of skeleton observations in the group treated with the highest doses of BIO (500 or 1000 mg/kg body wt/day) higher than those recorded in the vehicle-control group. In these cases, however, dose-effect relationships were unclear and a high incidence of the abnormality was also noted in the vehicle control group.

Study 2: In some sporadic cases, skeletal examination of this fetus revealed a small left eye socket; the presence of three femurs, tibia, and fibulas on the right side of the body (partially fused), and extra metatarsals, digits, and three sets of phalanges (four, five, and five) on the right hindlimbs.

Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Study 1: Except for a increase in the variational incidence of liver extra lobes, no consistent elevation in the occurrence of externally visible and soft-tissue (visceral) abnormalities was noted in the offspring of dams treated with the test chemical on GD 6-15.

Study 2: In some sporadic cases, slight dilation of the renal pelvis in one fetus from the control and one fetus from the 3 mg/kg/day dosage groups, and an aberrant umbilical artery in one and two fetuses at 3 and 10 mg/kg/day, respectively.

Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Study 1: At highest dose level tested (1000 mg/kg bw/day), the test chemical induced reduction of pregnancy weight gain during the treatment period (GD 6–15) accompanied by other clinical signs of toxicity and a decline in the ratio of pregnant to treated females. Thus, the NOAEL for the test chemical was attributed to 500 mg/kg bw/day.

Study 2: The NOAEL for the fetal/pups parameter was attributed to 30 mg/kg bw/day.

Study 3: The embryotoxic/teratogenic effects were observed in the highest tested dose group (1000 mg/kg bw/day).
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Remarks on result:
other: Not Specified
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Remarks on result:
other: Not Specified
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Remarks on result:
other: Not Specified
Abnormalities:
not specified
Developmental effects observed:
not specified
Treatment related:
not specified
Conclusions:
Study 1: Based on all the findings in the maternal and fetal parameters after the administration of the test chemical it was concluded that the NOAEL was 500 mg/kg bw/day for both maternal and fetal endpoints.

Study 2: Based on all the findings in the study, the NOAEL for the maternal and fetal/pups parameters wa found to be 30 mg/kg/bw/day.

Study 3: Based on all the findings in the study, it was observed that the No Observed Adverse Effect Level for the test chemical was found to 750 mg/kg bw/day and LOAEL was found to be 1000 mg/kg bw/day.
Executive summary:

Study 1:

The method in this publication was performed to evaluate any adverse effects on the maternal and fetal developmental parameters. In this study, the test chemical was administered through an oral gavage route in the dose concentrations of 0, 125, 250, 500 and 1000 mg/kg bw/day to Wistar rats. These rats were obtained fromOswaldo CruzFoundation Central Animal House breeding stock. The age of animals at the time they were received was about 60 days. The weight of the animals at the initiation of the study was approximately 200 grams. The rats were housed individually in standard plastic cages with stainles-steel cover lids and pinewood shavings as the bedding-material. A pelleted diet (Nuvital, for laboratory rats and mice) and filtered tap water was provide to ratsad libitum.The male and female animals were mated in ratio of 2 females to 1 male in the male animal's cage. The animals were cohabited for 2 hours and the presence of sperm in the vaginal smear was indicated as the day '0' of gestation. The doses at the concentration of125, 250, 500 and 1000 mg/kg bw/day were prepared in the vehicle, corn oil. The amount of the vehicle was3.75 g/kg body wt/day. These concentrations were administered to the mated dams daily during gestation day (GD) 6 to 15. The mated females were observed for mortality and clinical signs till GD 21. On GD 21, all the dams were sacrificed by CO2 asphyxiation. After maternal death confirmation,the gravid uterus was quickly removed and weighed with its contents. Ovaries were excised and cleared of adhering fat, and the number of corpora lutea graviditatis was counted. The uterine wall was carefully open and the number of living and dead fetuses and resorptions was counted. The method was employed to identify all sites of implantations including those that were completely resorbed. Briefly, the opened uterus was immersed in a 10% ammonium sulfate solution for 10 min, rinsed in running water and then immersed for additional 10 min in a solution containing equal parts of 2% potassium ferricyanide and 1% hydrochloric acid. Resorptions were classified as early resorption, if an implantation site does not correspond to a fetus or remains of embryo and placenta that are visible to the naked eye; intermediate resorption, if parts of the body of embryos and placentas cannot be differentiated macroscopically; or late resorption, if parts of embryo/fetus and placenta can be differentiated macroscopically. A dead fetus was a non live fetus with discernible digits in any or all paws. All living fetuses were weighed, examined under a stereomicroscope for externally visible abnormalities, and numbered with a marker pen. One-third of the fetuses from each litter-chosen at random-was fixed in Bouin's solution for further evaluation of soft tissue anomalies. Heart, thymus, liver, spleen, kidneys and lungs of sectioned fetuses were also weighed. The remaining fetuses were fixed in a 5% formalin solution, macerated in potassium hydroxide, cleared with a glycerin–KOH solution and stained with Alizarin Red S for skeletal evaluation Soft-tissue and skeleton abnormalities were recorded. After the examinations, it was observed that, all dams from control and test chemical treated groups survived to scheduled euthanasia on GD 21. Although, piloerection, vocalization and chromodacryorrhea were noted in rats treated with the highest dose of the test chemical (1000 mg/kg body wt/d). Only a few rats treated with the second highest dose of the test chemical (500 mg/kg body wt) presented red tears and one was hypoactive. No clinical signs was apparent after the period of treatment with the two highest doses and, at any time, among rats that received doses of the test chemical lower than 500 mg/kg body wt. Except for a decrease in weight gain of dams of the highest dose group (1000 mg/kg bw/day) during the treatment period (GD 6–15), pregnancy weight gain of rats exposed to the test chemical did not differ from that of vehicle-treated controls. The post mortem examinations did not reveal any changes in the organ weights and gross necropsy. In fetal parameters, the number of corpora lutea did not differ when compared with control groups. Similarly, the average litter size and the fetal body weights from all the dose groups did not significantly differ from the control groups. Although, he number of live offsprings were decreased at 1000 mg/kg bw/day. Also, the sex ratio was slightly inclined towards male than females in the highest tested dose groups. No consistent dose-related increase in the incidence of fetal skeleton anomalies was noted in the offspring of the test chemical treated dams. Only in a few instances (e.g., Axis misshapen, right and left Spine scapula misshapen) was the occurrence of skeleton observations in the group treated with the highest doses of BIO (500 or 1000 mg/kg body wt/day) higher than those recorded in the vehicle-control group. In these cases, however, dose-effect relationships were unclear and a high incidence of the abnormality was also noted in the vehicle control group. Also, except for a increase in the variational incidence of liver extra lobes, no consistent elevation in the occurrence of externally visible and soft-tissue (visceral) abnormalities was noted in the offspring of dams treated with the test chemical on GD 6-15. Thus, based on all the findings in the maternal and fetal parameters after the administration of the test chemical it was concluded that the NOAEL was 500 mg/kg bw/day for both maternal and fetal endpoints.

Study 2:

A study conducted in this publication was performed to evaluate the developmental effect of the test substance on Crl:CD(SD) IGS BR VAF/Plus rats. In this study, the rats were obtained from the Charles River Laboratories, Raleigh, NC. After a short acclimatization period, the rats were assigned to individual housing on the basis of computer-generated random units, except during the 5-day mating period when each pair of male and female rats was housed in the male rat’s cage. The healthy mated female rats, weighing 213 to 241 g, were assigned to four dosage groups, 25 rats/group, using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on the day when sperm was found in the vaginal smear or a copulatory plug was found in the vagina. The presence of spermatozoa and/or a copulatory plug in situ was designated as gestational day 0 (GD 0). The study room was independently supplied with at least 10 changes per hour of 100% fresh air passed through 99.97% HEPA filters. Environmental controls were set to maintain temperatures of 64◦F to 79◦F, with relative humidity of 30% to 70%; a 12:12- h light-dark lighting cycle was used. Certified Rodent DietR 5002 and reverse osmosis deionized water (with chlorine added to the processed water as a bacteriostat) were provided ad libitum to the rats. Oral dosages of 0, 3, 10, or 30

mg/kg/day alpha-iso-methylionone in corn oil were administered by gavage on gestational days 7 to 17. Rats were observed for viability, clinical signs, body weights, and feed consumption. Caesarean sectioning and necropsy occurred on gestational day 21. Uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. Numbers of corpora lutea were also recorded. Fetuses were weighed and examined for gender, gross external changes, and soft tissue or skeletal alterations. After all the evaluations, it was observed that, no maternal or fetal deaths occurred. No test chemical related clinical signs were observed. Feed consumption, body weight gains, gross tissue changes at necropsy, and caesarean section or litter parameters, as well as fetal developmental morphology, were unaffected by dosages of the test chemical till 30 mg/kg/day. Based on all the above findings, no observed adverse effect levels for maternal and developmental parameters were found to be greater than or equal to 30 mg/kg bw/day for the test chemical. Also, it was concluded that the test chemical was not developmental toxicant in rats at maternal doses of up to 30 mg/kg/day.

Study 3:

The method in the publication was used to evaluate the reproductive and developmental toxicity of the test chemical. In this method, the test chemical was administered into the rats by oral gavage in the concentrations of 0. 250, 500, 750 and 1000 mg/kg bw/day. The vehicle used to gavage the test chemical was corn oil. The source for the animals was Center for breeding of laboratory animals of the Oswaldo Cruz Foundation (Cecal-Fiocruz) and weighed around 200 -300 g at the initiation of the study. The rats were kept in the controlled temperature environment wherein the humidity was maintained around 70% and photoperiod was adjusted to 12 hours dark and 12 hours light. The rats were housed in standard plastic cages with stainless steel cover lids and wood shavings as bedding and kept under conditions of controlled temperature. The animals were mated in the ratio of 2:1 (2 females and 1 males) and were cohabited for 2 hours. The pregnancy was confirmed by the presence of sperm in the vaginal smear and was referred to as day '0' of pregnancy. On day 21 of gestation all dams were anesthetized and killed by CO2 inhalation. The gravid uterus was weighed with its contents and the number of implantation sites, living and dead fetuses, and resorptions was recorded.The ratio of resorptions per implantations, and the percentage of resorptions per implantations per litter were substantially increased, while the ratio of live fetuses per implantations per litter was drastically decreased, in dams treated with the highest dose of the test chemical (1000 mg/kg bw/day). In maternal animals, no signs of toxicity was observed till 750 mg/kg bw/day. The animals gained normal weight until sacrifice. After sacrifice and gross necropsy, the gravid uterus weight did not differ significantly till 750 mg/kg bw/day. Although, at 1000 mg/kg bw/day dose group, the number of resorptions were observed to be increased in maternal animals. Also, the gravid uterine weight was observed to be decreased significantly as compared to the control group. Also, the ratio of implantation sites to resorptions was found to be lower as compared to the control group. Embryolethality was much less pronounced with lower doses of the test chemical (250, 500 and 750 mg/kg). The findings thus suggested that the test chemical, at doses as high as 1000 mg/kg, killed most of the exposed embryos but, at lower doses (from 250 up to 750 mg/kg), which per se did not cause a high proportion of embryo death. No externally-visible anomaly was noted in fetuses from dams treated orally with the test chemical only on pregnancy day 11. Thus, based on all the findings in the study, it was observed that the No Observed Adverse Effect Level for the test chemical was found to 750 mg/kg bw/day and LOAEL was found to be 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data is from a Klimisch 2 database and provides a robust study summary.
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

Developmental toxicity

In different studies, Reaction mass the test chemical has been investigated for reproductive toxicity to a greater or lesser extent. Often are the studies based on in vivo experiments in rodents, i.e. most commonly in rats for the test chemical.

The method in this publication was performed to evaluate any adverse effects on the maternal and fetal developmental parameters. In this study, the test chemical was administered through an oral gavage route in the dose concentrations of 0, 125, 250, 500 and 1000 mg/kg bw/day to Wistar rats. These rats were obtained fromOswaldo CruzFoundation Central Animal House breeding stock. The age of animals at the time they were received was about 60 days. The weight of the animals at the initiation of the study was approximately 200 grams. The rats were housed individually in standard plastic cages with stainless-steel cover lids and pinewood shavings as the bedding-material. A pelleted diet (Nuvital, for laboratory rats and mice) and filtered tap water was provide to ratsad libitum.The male and female animals were mated in ratio of 2 females to 1 male in the male animal's cage. The animals were cohabited for 2 hours and the presence of sperm in the vaginal smear was indicated as the day '0' of gestation. The doses at the concentration of125, 250, 500 and 1000 mg/kg bw/day were prepared in the vehicle, corn oil. The amount of the vehicle was3.75 g/kg body wt/day. These concentrations were administered to the mated dams daily during gestation day (GD) 6 to 15. The mated females were observed for mortality and clinical signs till GD 21. On GD 21, all the dams were sacrificed by CO2 asphyxiation. After maternal death confirmation,the gravid uterus was quickly removed and weighed with its contents. Ovaries were excised and cleared of adhering fat, and the number of corpora lutea graviditatis was counted. The uterine wall was carefully open and the number of living and dead fetuses and resorptions was counted. The method was employed to identify all sites of implantations including those that were completely resorbed. Briefly, the opened uterus was immersed in a 10% ammonium sulfate solution for 10 min, rinsed in running water and then immersed for additional 10 min in a solution containing equal parts of 2% potassium ferricyanide and 1% hydrochloric acid. Resorptions were classified as early resorption, if an implantation site does not correspond to a fetus or remains of embryo and placenta that are visible to the naked eye; intermediate resorption, if parts of the body of embryos and placentas cannot be differentiated macroscopically; or late resorption, if parts of embryo/fetus and placenta can be differentiated macroscopically. A dead fetus was a non live fetus with discernible digits in any or all paws. All living fetuses were weighed, examined under a stereomicroscope for externally visible abnormalities, and numbered with a marker pen. One-third of the fetuses from each litter-chosen at random-was fixed in Bouin's solution for further evaluation of soft tissue anomalies. Heart, thymus, liver, spleen, kidneys and lungs of sectioned fetuses were also weighed. The remaining fetuses were fixed in a 5% formalin solution, macerated in potassium hydroxide, cleared with a glycerin–KOH solution and stained with Alizarin Red S for skeletal evaluation Soft-tissue and skeleton abnormalities were recorded. After the examinations, it was observed that, all dams from control and test chemical treated groups survived to scheduled euthanasia on GD 21. Although, piloerection, vocalization and chromodacryorrhea were noted in rats treated with the highest dose of the test chemical (1000 mg/kg body wt/d). Only a few rats treated with the second highest dose of the test chemical (500 mg/kg body wt) presented red tears and one was hypoactive. No clinical signs was apparent after the period of treatment with the two highest doses and, at any time, among rats that received doses of the test chemical lower than 500 mg/kg body wt. Except for a decrease in weight gain of dams of the highest dose group (1000 mg/kg bw/day) during the treatment period (GD 6–15), pregnancy weight gain of rats exposed to the test chemical did not differ from that of vehicle-treated controls. The post mortem examinations did not reveal any changes in the organ weights and gross necropsy. In fetal parameters, the number of corpora lutea did not differ when compared with control groups. Similarly, the average litter size and the fetal body weights from all the dose groups did not significantly differ from the control groups. Although, he number of live offsprings were decreased at 1000 mg/kg bw/day. Also, the sex ratio was slightly inclined towards male than females in the highest tested dose groups. No consistent dose-related increase in the incidence of fetal skeleton anomalies was noted in the offspring of the test chemical treated dams. Only in a few instances (e.g., Axis misshapen, right and left Spine scapula misshapen) was the occurrence of skeleton observations in the group treated with the highest doses of BIO (500 or 1000 mg/kg body wt/day) higher than those recorded in the vehicle-control group. In these cases, however, dose-effect relationships were unclear and a high incidence of the abnormality was also noted in the vehicle control group. Also, except for a increase in the variational incidence of liver extra lobes, no consistent elevation in the occurrence of externally visible and soft-tissue (visceral) abnormalities was noted in the offspring of dams treated with the test chemical on GD 6-15. Thus, based on all the findings in the maternal and fetal parameters after the administration of the test chemical it was concluded that the NOAEL was 500 mg/kg bw/day for both maternal and fetal endpoints.

A study conducted in this publication was performed to evaluate the developmental effect of the test substance on Crl:CD(SD) IGS BR VAF/Plus rats. In this study, the rats were obtained from the Charles River Laboratories, Raleigh, NC. After a short acclimatization period, the rats were assigned to individual housing on the basis of computer-generated random units, except during the 5-day mating period when each pair of male and female rats was housed in the male rat’s cage. The healthy mated female rats, weighing 213 to 241 g, were assigned to four dosage groups, 25 rats/group, using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on the day when sperm was found in the vaginal smear or a copulatory plug was found in the vagina. The presence of spermatozoa and/or a copulatory plug in situ was designated as gestational day 0 (GD 0). The study room was independently supplied with at least 10 changes per hour of 100% fresh air passed through 99.97% HEPA filters. Environmental controls were set to maintain temperatures of 64◦F to 79◦F, with relative humidity of 30% to 70%; a 12:12- h light-dark lighting cycle was used. Certified Rodent DietR 5002 and reverse osmosis deionized water (with chlorine added to the processed water as a bacteriostat) were provided ad libitum to the rats. Oral dosages of 0, 3, 10, or 30 mg/kg/day alpha-iso-methylionone in corn oil were administered by gavage on gestational days 7 to 17. Rats were observed for viability, clinical signs, body weights, and feed consumption. Caesarean sectioning and necropsy occurred on gestational day 21. Uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. Numbers of corpora lutea were also recorded. Fetuses were weighed and examined for gender, gross external changes, and soft tissue or skeletal alterations. After all the evaluations, it was observed that, no maternal or fetal deaths occurred. No test chemical related clinical signs were observed. Feed consumption, body weight gains, gross tissue changes at necropsy, and caesarean section or litter parameters, as well as fetal developmental morphology, were unaffected by dosages of the test chemical till 30 mg/kg/day. Based on all the above findings, no observed adverse effect levels for maternal and developmental parameters were found to be greater than or equal to 30 mg/kg bw/day for the test chemical. Also, it was concluded that the test chemical was not developmental toxicant in rats at maternal doses of up to 30 mg/kg/day.

The method in the publication was used to evaluate the reproductive and developmental toxicity of the test chemical. In this method, the test chemical was administered into the rats by oral gavage in the concentrations of 0. 250, 500, 750 and 1000 mg/kg bw/day. The vehicle used to gavage the test chemical was corn oil. The source for the animals was Center for breeding of laboratory animals of the Oswaldo Cruz Foundation (Cecal-Fiocruz) and weighed around 200 -300 g at the initiation of the study. The rats were kept in the controlled temperature environment wherein the humidity was maintained around 70% and photoperiod was adjusted to 12 hours dark and 12 hours light. The rats were housed in standard plastic cages with stainless steel cover lids and wood shavings as bedding and kept under conditions of controlled temperature. The animals were mated in the ratio of 2:1 (2 females and 1 males) and were cohabited for 2 hours. The pregnancy was confirmed by the presence of sperm in the vaginal smear and was referred to as day '0' of pregnancy. On day 21 of gestation all dams were anesthetized and killed by CO2 inhalation. The gravid uterus was weighed with its contents and the number of implantation sites, living and dead fetuses, and resorptions was recorded.The ratio of resorptions per implantations, and the percentage of resorptions per implantations per litter were substantially increased, while the ratio of live fetuses per implantations per litter was drastically decreased, in dams treated with the highest dose of the test chemical (1000 mg/kg bw/day). In maternal animals, no signs of toxicity was observed till 750 mg/kg bw/day. The animals gained normal weight until sacrifice. After sacrifice and gross necropsy, the gravid uterus weight did not differ significantly till 750 mg/kg bw/day. Although, at 1000 mg/kg bw/day dose group, the number of resorptions were observed to be increased in maternal animals. Also, the gravid uterine weight was observed to be decreased significantly as compared to the control group. Also, the ratio of implantation sites to resorptions was found to be lower as compared to the control group. Embryolethality was much less pronounced with lower doses of the test chemical (250, 500 and 750 mg/kg). The findings thus suggested that the test chemical, at doses as high as 1000 mg/kg, killed most of the exposed embryos but, at lower doses (from 250 up to 750 mg/kg), which per se did not cause a high proportion of embryo death. No externally-visible anomaly was noted in fetuses from dams treated orally with the test chemical only on pregnancy day 11. Thus, based on all the findings in the study, it was observed that the No Observed Adverse Effect Level for the test chemical was found to 750 mg/kg bw/day and LOAEL was found to be 1000 mg/kg bw/day

Justification for classification or non-classification

Thus,comparing this value with the criteria of CLP regulation ionone (8013-90-9) can be "Not classified" for reproductive and developmental toxicity.