5-methylheptan-3-one oxime

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

yes At arrival, the body weight range of females was 184 to 215 g instead of 175 to 200 g as specifi ed in the Study Protocol.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5-methylheptan-3-one oxime

EC Number:

245-010-8

EC Name:

5-methylheptan-3-one oxime

Cas Number:

22457-23-4

Molecular formula:

C8H17NO

IUPAC Name:

5-methylheptan-3-one oxime

impurity 1

Reference substance name:

(E)-3,4-dimethylhexan-2-one oxime

IUPAC Name:

(E)-3,4-dimethylhexan-2-one oxime

impurity 2

Reference substance name:

(Z)-3,4-dimethylhexan-2-one oxime

IUPAC Name:

(Z)-3,4-dimethylhexan-2-one oxime

Test material form:

liquid

Specific details on test material used for the study:

The test item will be Stemone.
The following information refers to the original batch of test item received for the study:
Batch Number : PE00160768
Date of expiry : 02 December 2018
Appearance : Colourless to pale yellow, clear liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage conditions : +4°C protected from light
Density : 0.88797 g/mL

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Assessment of the formulation for stability, homogeneity and concentration are regarded as unnecessary, the test item and control item being used in the form supplied.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

A total of 122 Hsd: Sprague Dawley SD rats (57 males and 65 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, will be ordered from Charles River Italia S.p.A., Calco (Lecco), Italy.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy.
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: 200 to 225 g
- Fasting period before study:
- Housing: Clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and
floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese.). Each cage tray held absorbent material.
- Diet (e.g. ad libitum): A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via
G. Galilei, 4, 20019, SettimoMilanese (MI), Italy) was offered ad libitum throughout the study
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: An acclimatisation period of 21

DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the
effect of the test item was present in the drinking water or the diet. Records of analyses of water and
diet are kept on file at RTC.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±2°C
- Humidity (%): 55%±15%
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): Rooms were lit by artificial light for 12 hours each day.

Administration / exposure

Route of administration:

dermal

Vehicle:

corn oil

Details on exposure:

Before the start of treatment, fur was removed from the dorsal surfaces of the trunk over an estimat ed area of at least 10% of the total body surface (approximately 5 x 7 cm) using an electric clipper with a suitable blade. Care was taken to avoid any damage or abrasion to the skin. The clipping procedure was repeated as necessary during the course of the study to keep the area free of fur.
The test item or control item was applied uniformly over the prepared skin area of the animals. Control animals received the control item at the highest dose level. The dose was administered to each animal on the basis of the most recently recorded body weight and the amount administered was recorded for each animal. The treated area was covered using a piece of porous gauze covered with a self-adherent bandage. An Elizabethan collar was placed around the neck of each animal to prevent grooming of the treated site and oral ingestion of the test item or control item following dosing. The Elizabethan collars remained in place for approximately 6 hours following dosing.
After a period of approximately 6 hours, the Elizabethan collar and the bandagewere removed.
The treated skin site was then gently washed free of any remaining test item using 0.9% physiological saline.

Details on mating procedure:

Mating - (Main groups)
Pairing will be monogamous (one male to one female). A vaginal smear will be taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female will be paired with the same male until positive identification occurs or 14 days have elapsed.

Parturition and gestation length - (Main groups)
A parturition check will be performed from Day 20 to Day 25 post coitum.
Females which do not give birth after 25 days of post coitum period will be sacrificed shortly after.
Gestation length will be calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth when the parturition is defined complete (Day 0 post partum).

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Formulation analyses were regarded as unnecessary, the test item being used as supplied.

Duration of treatment / exposure:

Males
The amount of test item or control item will be adjusted once per week for each animal according to the last recorded body weight.
Animals will be dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, thereafter during pairing until Day 19 post coitum included. Dosing will be re-initiated starting from Day 4 post partum until Day 13 post partum or the day before sacrifice.
Females
The amount of test item or control item will be adjusted once per week for each animal according to the last recorded body weight.
During the gestation period, the amount will be calculated according to individual body weight on Days 0, 7, 14 and 19 post coitum and Days 4 and 7 post partum. Thereafter individual dose volumes will remain constant.

Frequency of treatment:

Daily.

Details on study schedule:

Each main group will comprise 10 male and 10 female rats (Groups 1 to 4).
Two groups (control and high dose levels) will include 5 animals per sex to be sacrificed after 2 weeks of recovery (Groups 5 and 6).

No. of animals per sex per dose:

Each main group will comprise 10 male and 10 female rats (Groups 1 to 4).
Two groups (control and high dose levels) will include 5 animals per sex to be sacrificed after 2 weeks of recovery (Groups 5 and 6).

Control animals:

yes

Details on study design:

Individuals will be uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed and individually.
The cages will be identified by a label and recording the study number, animal numbers and details of treatment.
The arrangement of cages in batteries will be such that cages from each treatment group will be evenly distributed across the battery to minimise possible environmental effects.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: At least once daily during the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study,
each animal was observed and any clinical signs were recorded.

BODY WEIGHT: Yes
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 19 post coitum.
Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.

FOOD EFFICIENCY:
The weight of food consumed by each cage ofmales and females was recorded weekly during the
pre-mating period starting from allocation. Individual food consumption for the females
was measured on Days 7, 14 and 19 post coitum starting from Day 0 post coitum and on Days 4, 7
and 13 post partum starting from Day 1 post partum.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes
Dosing phase:
Leucocytosis was recorded in some males from all treated groups, such as: animal nos. X0450032 (200mg/kg/day), X0450058 (500mg/kg/day), X0450064 and X0450070 (1000mg/kg/day)..
Compared with mean control data, individual increases were 1.6 to 2.3 fold, with no clear dose-relation.

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli); and for assessment of grip strength. For the females, the tests were performed on Day 13 post partum. Measurements were performed using a computer generated random order (for the main groups).
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. For the females, the tests were performed on Day 13 post partum. Measurements were performed using a computer generated random order (for the main groups). Once duringWeek 2 of recovery, the motor activity was also measured for all
animals.

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

Females will be evaluated for oestrous cyclicity at least 7 days before allocation to groups up to the start of dosing and animals that exhibit irregular cycle will not be included in the study.
Vaginal smears will be taken daily in the morning starting two weeks before pairing throughout the mating period until a positive identification of copulation is made. The vaginal smear data will be examined to determine the following:
a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears will also be taken from all females, before dispatch to necropsy.

Sperm parameters (parental animals):

No

Litter observations:

As soon as possible, after parturition is considered complete (Day 0 post partum), all pups (live and dead) will be counted, sexed and live pups will be identified.
Live pups will be individually weighed on Days 1, 4, 7 and 13 post partum.
Pups killed or dying during the lactation period will be weighed before the despatch to necropsy.
Observation will be performed once daily for all litters.

Postmortem examinations (parental animals):

Parental animals in extremis, killed for humane reasons and those that have completed the scheduled test period and selected for blood collection, will be killed by exsanguination under isofluorane anaesthesia.
Parental males (Main groups)
The males will be killed after the mating of all females or after at least 28 days of treatment period.
Parental females (Main groups):
The females with total litter loss will be killed on the day of the occurrence of total litter loss or shortly after.
The females showing no evidence of copulation will be killed after 25 days of the last day of the mating session (see section 4.2.10).
The females which do not give birth 25 days after positive identification of mating will be killed shortly after (see section 4.2.11).

Postmortem examinations (offspring):

Pups killed for humane reasons or those that have completed the scheduled test period (Day 4 post partum or Day 14 post partum) will be euthanised by intraperitoneal injection of Sodium Thiopenthal.
Pups selected for blood collection for hormone determination will be killed on the day of blood sampling (see section 4.3.4)

Statistics:

Standard deviations will be calculated as appropriate. For continuous variables the significance of the differences amongst group means will be assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings will be carried out by means of the non-parametric Kolmogorov-Smirnov test if n is more than 5.
The non-parametric Kruskal-Wallis analysis of variance will be used for the other parameters. Intergroup differences between the control and treated groups will be assessed by the non-parametric version of the Williams test. The criterion for statistical significance will be p<0.05.
Further tests will be used as considered appropriate. Details of all tests used and the data to which they are applied will be included in the Final Report.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

The treatment site was observed daily. During treatment, the observations were performed twice daily (at the end of the treatment period, after the removal of the bandage and approximately 24 hours a fter treatment start).
No signs were observed in control animals of both sexes during the whole study.

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

Dose level of 200mg/kg/day:
Two males (nos: X0450024 and X045030) showed a slight desquamation on one occasion duringWeek 4 of treatment. Male no. X0450028 showed slight erythema from Day 5 to Day 14 of treatment. Skin thickening was present in all males from Day 17 of mating (Week 4 of treatment). In females, a slight erythema was present in one animal (no. X0450037) on Day 7 of treatment only. During the post coitum period, when animals were treated (from Day 0 to Day 19), slight to moderate erythema was described in one female (no. X0450033) for a few days. Skin thickening was present in 9/10 females, starting to be evident from Day 12 or 14 post coitum. From Day 20 post coitum to Day 3 post partum (when treatment stopped), skin thickening was still observed in 1/10 females (no. X0450037) on Day 20 post coitum. When treatment started (from Day 4 to Day 13 post partum), abrasion and/or scabs and/or desquamation were evident in 1/10 females (no. X0450031) starting from Day 8 post partum. For both males and females, complete recovery from the signs was noted approximately 16 hours after the removal of the bandage.

Dose level of 500mg/kg/day:
Erythema and/or desquamation were present in 7 out of 10 males. Erythema started to be evident from Day 5 of dosing and desquamation from Day 21 of mating (Week 4 of treatment). Slight to moderate skin thickening was recorded in all males and started to be evident on Day 17 of mating (Week 4 of treatment). Scabs and/or hairloss were observed fromWeek 1 of treatment up to Day 7 post coitum in one female (no. X0450049). Slight to moderate erythema was observed in 4 females (nos.
X0450045, X0450047, X0450055, X0450059) and was evident on Day 8 of treatment up to Day 15 of treatment. Slight and/or moderate erythema was still observed in 1 female (no. X0450059) from Day 4 to 6 post coitum. Complete recovery, except for skin thickening, both for males and females was still observed after approximately 16 hours from the removal of the bandage (before the next treatment).

Dose level of 1000mg/kg/day:
total of 9 out of 10 males showed slight erythema fromWeek 1 of treatment and desquamation (all animals) during last week of treatment. Moderate skin thickening was recorded in all males and started to be evident on Day 17 of mating phase (Week 4 of treatment). Scabs were noted in 3 males fromWeek 2 of treatment on occasion. Fissuration was noted in 3 males during the first 2 weeks of treament. Slight to moderate erythema was observed in 9/10 females during the first 2 weeks of treatment. In addition, fissuration was evident from Day 8 up to Day 12-13 of treatment in 4/10 females. During the post coitum period, a slight to moderate erythema was recorded in 1/10 females. In addition, 4/10 females showed scabs and 2 of these also desquamation for a few days. During the discontinuation of treatment (from Day 20 post coitum to Day 3 post partum), the only sign was desquamation, observed in 2 females. From Day 4 post partum to Day 13 post partum, when the treatment started again, the following signs were noted: abrasion in 1 female, desquamation in 4 females and hairloss in 2 females. Complete recovery of the signs was evident after approximately 16 hours from the removal of the bandage (before the next treatment).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male of the recovery group receiving 1000mg/kg/day was found dead on Day 27 of the study (no. X0450096). Macroscopically, the findings observed in this animal included: distended caecum, red colour or areas of jejunum, mesenteric lymph nodes and thymus. At histopathology, inflammatory cell foci of heart and liver or glandular dilatation of the stomach were noted. Such findings did not establish the factors contributory to the death.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Main and Recovery groups
No differences were seen in body weight of treated animals when compared to controls, both in the main and recovery groups. The only difference noted in body weight gain of the main groups was evident in the high dose males (1000mg/kg/day) on Day 8 of the study and in low dose females (200mg/kg/day) on Day 7 post partum. The differences were considered incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Main and Recovery groups
Food consumption was unaffected by treatment in both sexes during the study.

Food efficiency:

no effects observed

Description (incidence and severity):

Main and Recovery groups
Food efficiency was unaffected by treatment in both sexes during the study.

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

no effects observed

Description (incidence and severity):

Main and Recovery groups
Water consumption was unaffected by treatment in both sexes during the study.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dosing phase
Leucocytosis was recorded in some males from all treated groups, such as: animal nos. X0450032 (200mg/kg/day), X0450058 (500mg/kg/day), X0450064 and X0450070 (1000mg/kg/day). Compared with mean control data, individual increases were 1.6 to 2.3 fold, with no clear dose-relation.

Recovery phase
No changes of the white blood cells were recorded in treated males, confirming complete reversibility. The statistically significant difference of platelets recorded between controls and treated males was not recorded at the dosing phase, therefore it was considered unrelated to treatment.

Coagulation - Dosing phase
No relevant changes were recorded.

Coagulation - Recovery phase
No relevant changes were recorded.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups - Dosing phase
Phosphorus was increased in males dosed at 500 and 1000mg/kg/day. Mean group data were 16% and 19%, respectively, above controls. Since changes were of slight severity and there were no other related findings, these increases were considered of no toxicological importance. The other statistically significant differences recorded between control and treated females (decreases of creatinine and phosphorus in females) were recorded in the low and/or intermediate groups only, therefore they were considered incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Main groups
Motor activity recorded at the end of the treatment period was comparable between control and treated main groups. Statistically significant increase in landing foot splay measurement (individual and mean data for first and second trials) was noted in treated males of Group 4 (1000mg/kg/day) when compared to the control group. The variation was considered of no toxicological significance, since it was observed in only one sex.

Recovery groups
A variation (increase) was noted in the motor activity of females, both at the end of treatment and recovery periods. This change was considered of no toxicological significance, since it was not consistent with that observed in the main female group at the same dose level. Moreover, an increase in the landfoot splay (second trial) was noted at the end of the recovery period in high dose females (1000mg/kg/day), when compared to the control group. However, due to the inconsistent presence of the changes between sexes and considering that they occurred at the end of the recovery phase, this variation was considered of no toxicological relevance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

An increased severity of hyaline droplets accumulation in kidneys from moderate to marked degree was observed in high dose males, when compared with controls. However, since the increase in hyaline droplets in the kidneys is not relevant for human, it was decided not to proceed with the extension of histopathological examination.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

No relevant changes were recorded in terminal body weight and organ weights (absolute and relative) in animals of both sexes, when compared to the control group. At macroscopic observations, no treatment-related changes were noted.
At microscopic observations changes were noted in the kidneys of high dose males, when compared with controls. An increased severity of hyaline droplets accumulation in kidneys from moderate to marked degree was observed in high dose males.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All animals mated in approximately 4 days (pre-coital interval) with the exception of one male which copulated after 7 days of cohabitation. The number of copulatory plugs found in the cage was similar between control and treated animals.
Oestrous cycle and copulatory index were similar between treated and control animals. Fertility index both for males and females was 100% each in the control and Group 3 and 90% in Groups 2 and 4. One male, each in Groups 2 (no. X0450036) and 4 (no. X0450072) failed to induce pregnancy. These isolated cases were considered incidental.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Reproductive performance:

no effects observed

Description (incidence and severity):

Gestation periods were similar in treated groups and controls. All dams gave birth between
Day 22 post coitum (mean value). Corpora lutea, implantations, total litter size at birth and
pre-natal loss data (percentage) were similar in control and treated groups.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

female reproductive system

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Cold to touch, apparently no food intake (milk) and small appearance were in general noted in control and treated groups. Wound on scapular region and hairloss were observed in midand high dose pups. No toxicological significance was attributed to these findings which are commonly observed in the litters during the lactation period.

Dermal irritation (if dermal study):

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No abnormalities were recorded at the external and internal examination of pups found dead, in culled pups at Day 4 post partum or in pups sacrificed with dam on Day 14 post partum.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No differences were noted in thyroid weight between control and treated pups.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Litter data and sex ratios were unaffected by treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No relevant changes were recorded in terminal body weight and organ weights (absolute and relative) in animals of both sexes, when compared to the control group.
At macroscopic observations, no treatment-related changes were noted.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

sexual maturation

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

female reproductive system

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and reproductive and developmental toxicity was considered to be 1000mg/kg/day for both males and females.

Executive summary:

The NOAEL is 1000 mg/kg/day.