4-methylpentan-2-one oxime

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not indicated

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

not specified

Remarks:

No statement on GLP compliance is mentioned in the publication.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-methylpentan-2-one oxime

EC Number:

203-298-2

EC Name:

4-methylpentan-2-one oxime

Cas Number:

105-44-2

Molecular formula:

C6H13NO

IUPAC Name:

4-methylpentan-2-one oxime

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: methyl isobutyl ketoxime (MIBKO); CAS number 105-44-2
- Source and lot/batch No.of test material: supplied by Honeywell International / batch 061001.
- Expiration date of the lot/batch: no data
- Purity test date: no data
- Purity: 99.9%
- Appearance: clear, colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: no data
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no data
- Preliminary purification step (if any): no data
- Final dilution of a dissolved solid, stock liquid or gel: no data
- Final preparation of a solid: no data

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: (P) approx. 5 wks; (F1) 3 wks
- Age at start of treatment: approx. 6 weeks
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: Cages were solid polycarbonate with Lignocel ¾ wood flakes for bedding. Animals were housed 4 to a cage except during mating, gestation, and lactation. During mating, 1 male and 1 female from the same treatment group were housed together. During gestation and lactation, females were housed individually or with their litters.
- Diet (e.g. ad libitum): standard rodent diet (SDS VRF1 Certified diet)
- Water (e.g. ad libitum): no data
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 40–70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-hour light-dark cycle

IN-LIFE DATES: no data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 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): 2 mL/kg
- Lot/batch no. (if required): No data
- Purity: No data

Details on mating procedure:

- M/F ratio per cage: During mating, 1 male and 1 female from the same treatment group were housed together until there were positive signs of copulation
- Length of cohabitation: during mating: 10 weeks
- Proof of pregnancy: not described
- Replacement of first male by another male with proven fertility after some days of unsuccessful pairing: not described
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: ?

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis at selected times indicated the test material formulations were stable and homogenous.

Duration of treatment / exposure:

Males: 10 weeks prior to mating and 2 weeks of mating (or until signs of mating were noted)
Females: 10 weeks prior to mating and 2 weeks of mating (or until signs of mating were noted), and through gestation and lactation
Selected pups were dosed by oral gavage from weaning until approx. 7 weeks of age (from PND 21 to PND 55)

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until [...] weeks after selected from the F1 litters: not applicable.
- Selection of parents from F1 generation when pups were [...] days of age: not applicable.
- Age at mating of the mated animals in the study (F0): 6+10+2=18 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

28 males and 28 females; 4 groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not described

Positive control:

no

Examinations

Parental animals: Observations and examinations:

Dams were observed three times per day starting on day 20 of gestation until parturition.

CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily
- Parameters: general signs of health status

BODY WEIGHT: Yes
- Time schedule for examinations:
• Males: weekly from the start of the study until sacrifice
• Females: weekly during the premating and mating periods, on gestation days 0, 3, 6, 10, 14, 17, and 21 and after parturition on postnatal days 1, 4, 7, 10, 14, 18 and 21. Nonmated females were weighed weekly until sacrifice.

FOOD CONSUMPTION: Food consumption was measured at the same frequency as the body weights.

Oestrous cyclicity (parental animals):

Three weeks prior to mating, vaginal smears were prepared to observe the estrus cycle of the F0 and F1 females.

Sperm parameters (parental animals):

not examined

Litter observations:

STANDARDISATION OF LITTERS
- Following delivery, the number of viable pups was determined, and the ratio of male to female pups was calculated
- Performed on day 4 postpartum: yes
- on PN day 4, litters were culled to 8 or 10 pups, with equal number of males and females where possible, by random selection.
ossible); excess pups were killed and discarded.

PARAMETERS EXAMINED
- Males were examined daily from day 38 until blanoreputial separation occurred
- Selected females were examined from day 28 until the vaginal opening occurred

The following parameters were examined in F1 offspring:
- Total litter size, number of male and female pups, number of stillborn and live births and grossly malformed pups, and pups showing abnormalities were recorded on postnatal day 1.
- The number of live and dead pups, as well as pups showing malformations or abnormalities, was recorded on postnatal days 4, 7, 14, 18 and 21.
- Litters were examined daily for dead pups.
- Body weight: Pups were also weighed on postnatal days 1, 4, 7, 11, 14, 18, 21, 25, and 28, as well as blanopreputial separation (males) and vaginal opening (females) occurred. Body weights for both males and females were recorded at blanoreputial separation or vaginal opening

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were killed following mating.
- Maternal animals: All surviving animals were sacrificed after weaning of the pups and subjected to a complete necropsy.
- all surviving male and female parent rats were sacrificed by exsanguination from the abdominal aorta under CO2/O2 anesthesia.

GROSS NECROPSY
During necropsy examination, the following organs were collected and preserved:
vagina, ovaries, uterus with cervix (after counting the number of implantation sites), testes, epididymides, seminal vesicles, prostate, brain, liver, kidneys, spleen, pituitary, and grossly abnormal organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Sections of the spleen and kidneys were examined microscopically.
- Examination of the other tissues was limited to the high dose and control animals. In addition, reproductive organs from all animals that failed to mate successfully were examined.

Postmortem examinations (offspring):

SACRIFICE
Subsequently, the pups were sacrificed until blanopreputial separation occurred (males)or until the vaginal opening occurred (females) or, if these events did not occur, held until approximately 8 weeks of age and then sacrificed.

GROSS NECROPSY
A complete necropsy was conducted on pups examined for sexual maturation.
All stillborn pups, pups found dead, or pups terminated in a moribund condition were examined macroscopically for structural abnormalities and pathological changes.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organs showing macroscopic changes were preserved in an aqueous-phosphate-buffered 4% solution of formaldehyde and subsequently examined microscopically.

Statistics:

Statistical analyses were performed by using one or multiple methods, as appropriate.
- For categorical data, Fisher’s exact probability test was used.
- For continuous data, one-way analysis of variance (ANOVA), followed by either Dunnett’s multiple comparison or Barlett’s test, was used. Kruskal-Wallis nonparametric ANOVA, followed by the Mann-Whitney U-test, was used for nonparametric data.
The following additional statistical evaluations were performed: Williams test for parametric monotone trend, Shirley’s test for nonparametric monotonic trend, and Steel’s test, if the dose response was not monotone. In addition, postimplantation loss and sex ratio were analyzed by the mixed linear model Wald chi-square test.

Reproductive indices:

To assess fertility and reproductive performance, the following data were collected:
number of males, mated with females, number of successful copulations, time to successful copulation, number of females with live-born pups, number of females with only stillborn pups, number of live pups at various intervals postpartum, number of pups lost, number of litters lost, total number of implantation sites, number of lost implantations, and the litter size.

From these data, the following parameters were calculated;
precoital time, duration of gestation, mating index, male fertility index, female fertility index, female fecundity index, gestation index, live birth index, pup survival and mortality until sacrifice, sex ratio, number of lost implantations, and postimplantation survival index.

Offspring viability indices:

live birth index, pup survival and mortality until sacrifice, sex ratio, number of lost implantations, and postimplantation survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation and chin rubbing were the only clinical signs attributed to administration of the test article.
These effects were observed in 100-mg/kg group males and females and diminished following 4 weeks of treatment.
No animals were found in moribund condition.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- One male at 30 mg/kg was found dead on day 32, and another male at 100 mg/kg was found dead on day 112.
- One control female was found dead on day 35.
The cause of death was not determined.

- One female was sacrificed for welfare reasons on day 1 of lactation. Clinical signs included piloerection, hunched posture, skin pallor, and hair loss. Necropsy observations included pale, inactive mammary tissue, pale liver, ovaries, pituitary, thyroid, enlarged spleen, and compacted material in the rectum. Neither of these deaths was considered to be treatment related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body-weight change were similar to controls at all time points.
Differences were not seen in terminal body weights in the parental animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was similar to controls at all time points.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No histological lesions were observed in the reproductive organs.
Minimal congestion was also observed in the spleens of 20 of 27 males and 19 of 26 females in the 100 mg/kg group.
Minimal to moderate hemosiderosis was observed in higher incidence and severity in the 100-mg/kg group males and females.
In the kidneys of male rats, cortical tubules with hyaline droplets were observed with increasing incidence and intensity in a dose-dependent manner. The minor changes observed in male kidneys at 30 mg/kg was not considered to be significant.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effects on estrus cycle were observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Details are described in Table 1.
- Mating: All but 1 female in the control group mated.
- No effects on precoital time, the number of pregnant females, fecundity, or duration of gestation were seen.
- No changes were observed in the timing of sexual maturation of F1 pups (i.e., completion of vaginal opening or preputial separation).
- The total number of pups delivered, number of stillborn pups, pup mortality, and pup weights were similar for all groups.
- One female each from the control, 10-mg/kg, and 100-mg/kg groups were not pregnant.
- One female at 30 mg/kg/day had a total litter loss with no pups born alive. No remarkable clinical signs were observed, but at necropsy revealed pale, inactive mammary tissue and fetal material in the stomach.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

spleen

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The total number of pups delivered, number of stillborn pups, pup mortality, and pup weights were similar for all groups. More details are described in table 1.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

No changes were observed in the timing of sexual maturation of F1 pups (i.e., completion of vaginal opening or preputial separation).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increases in spleen weight were observed in the 100-mg/kg group pups at day 28 of age, but were similar to controls in those animals exposed until they were 7 weeks of age.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: reproductive indices

Parameter	Control	10 mg/kg	30 mg/kg	100 mg/kg
No. of mating pairs	27	28	28	28
Female mating index	100	100	100	100
Female fertility index	96	96	100	96
Gestational index	100	100	100	100
No. of live litters (PN day 0)	26	27	28	27
No. of live litters (PN day 21)	26	27	27	26
Gestational length (days)	22.7	22.6	22.6	22.6
No. of implantation sites/litter	15.2	15.6	16.1	15.2
No. of total pups/litter	14.7	14.9	15.3	15.3
No. of live pups/litter (PN day 0)	14.5	14.6	14.9	15.1
Postimplantation survival index	93.1	92.2	93.6	95.0
No. of stillborn pups	7	8	12	9
Live birth index	98.2	98.1	97.3	98.9
Pup mortality (PN days 0 -4 precull)	4	4	5	8
Pup mortality (PN days 4 -21 postcull)	0	2	2	2

postimplantation survival index = (total number of offspring born/total number uterine implantation site) x 100; live birth index = number offspring on day 1 after littering/total number offspring born x 100; PN = postnatal

Table 2 Selected organ and relative organ weights (g/kg body weight)

	Males				Females
Parameter	Control	10 mg/kg	30 mg/kg	100 mg/kg	Control	10 mg/kg	30 mg/kg	100 mg/kg
Terminal body weight	590	602	593	598	325	329	325	330
Spleen weight	0.844	0.866	0.966	1.608@	0.605	0.629	0.615	0.871@
Relative spleen weight	0.144	0.144	0.163	0.269@	0.187	0.191	0.191	0.263##
Liver weight	23.18	24.99	24.87	25.67	14.97	15.97	15.81	16.36##
Relative liver weight	3.93	4.16	4.21	4.29#	4.64	4.84	4.87	4.97##
Kidney weight	4.06	4.25	4.31	4.37##	2.52	2.6	2.63	2.73##
Relative kidney weight	0.690	0.710	0.728#	0.731#	0.778	0.790	0.810	0.822

statistical key : analysis of variance and Dunnett's test: *p<0.05; **p<0.01; ***p<0.001; Williams test: #p<0.5; ##p<0.01; Shirley test: @p<0.01

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, exposure to the test substance does not result in reproductive toxicity or adverse effects in the F1 generation. The test substance is considered not classified as reproductive toxicant according to CLP Regulation.