4-methylpentan-2-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented, according to accepted guidelines

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: (F0) 46 days; (F1) 49 days
- Weight at study initiation: (F0) Males: 259-552 g; Females: 235-371 g; (F1) Males: 362-538 g; Females: 218-339 g
- Housing: individually is suspended wire-mesh cages
- Diet (e.g. ad libitum): Standard rodent chow ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 23 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes per hour: 12 to 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 3, 1998 To: May 1999

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2.0 m3 stainless steel and glass whole body inhalation chamber
- Method of holding animals in test chamber: cage
- Source and rate of air: not reported
- Method of conditioning air:not reported
- System of generating particulates/aerosols:not reported
- Temperature, humidity, pressure in air chamber: 22 ± 2°C and 30 to 70%, respectively
- Air flow rate: 12 to 15 air changes/hour
- Air change rate: 12 to 15 /hour
- Method of particle size determination: not reported
- Treatment of exhaust air:not reported

TEST ATMOSPHERE
- Brief description of analytical method used: measured 9 to 10 times during each daily exposure by a validated gas chromatographic method
- Samples taken from breathing zone: yes

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: copulatory plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Yes. Air in exposure chambers was sampled approximately every 35 minutes during exposure, and MIBK concentration verified using gas chromatography.

Duration of treatment / exposure:

F0 and F1 males: =70 days prior to mating until 1 day prior to euthanasia
F0 and F1 females: =70 days prior to mating until gestational day 20; PND 5 until 1 day prior to euthanasia
Exposure for F1 animals began on PND 22.
F2 animals were potentially exposed in utero and during PND 0 to 21 (but were not exposed in exposure chambers).

Frequency of treatment:

6 hours/day, 7 days/week

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 4 days of age.
- Age at mating of the mated animals in the study: 13 weeks

Remarks:

Doses / Concentrations:
500, 1000, or 2000 ppm
Basis:
other: target concentrations

Remarks:

Doses / Concentrations:
491, 999, and 1996 ppm
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
2012, 4093 or 8178 mg/m3
Basis:
analytical conc.
F0 generation

Remarks:

Doses / Concentrations:
2073, 4105 or 8219 mg/m3
Basis:
analytical conc.
F1 generation

No. of animals per sex per dose:

30

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: on the basis of results of previous studies using MIBK

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations: weekly

Oestrous cyclicity (parental animals):

To determine the estrous stage of each female F0 and F1, vaginal smears were prepared daily beginning 21 days before pairing and continuing until evidence of mating was present or the end of the mating period. The estrous stage of each female was also determined on the day of euthanasia.

Sperm parameters (parental animals):

Sperm motility and morphology for each F0 and F1 male was determined immediately following euthanasia. Homogenization-resistant spermatid and sperm production rates were also determined.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4 sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, and physical abnormalities


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- All surviving F0 animals were euthanized upon selection of the F1 generation, and all surviving F1 animals were euthanized following weaning of the F2 generation


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues/organs were prepared for microscopic examination: adrenals (2), aorta, bone with marrow (sternebrae), brain (forebrain, midbrain, hindbrain), coagulating gland, eyes with optic nerve (2), gastrointestinal tract (esophagus, stomach, duodenum, ileum, jejunum, cecum, colon, rectum), heart, kidneys (2), liver (sections of 2 lobes), lungs (including bronchi, fixed by inflation with fixative), lymph node (mesenteric), ovaries and oviduct, pancreas, peripheral nerve (sciatic), pituitary, prostate, salivary gland (submaxillary; 2), seminal vesicles (2), skeletal muscle (vastus medialis), skin with mammary gland, spinal cord (cervical), spleen, testes with epididymis (1) and vas deferens, thymus, thyroids (with parathyroids if present; 2), trachea, urinary bladder, uterus with vagina, and all gross lesions; in addition the following organs were weighed: adrenals, brain, epididymis (total and cauda), kidneys, liver, ovaries, pituitary, prostate, seminal vesicles with coagulating glands (with accessory fluids), spleen, testes, thymus, and uterus with oviducts and cervix.

Microscopic evaluations of the following tissues were performed for parental (10/sex/group) animals who were found dead or euthanized due to morbidity: adrenals, brain, epididymides (right; caput, corpus, and cauda), cervix, coagulating gland, kidneys, liver, lung, ovaries, oviducts, pituitary, prostate, seminal vesicles, spleen, testes (right), thymus, uterus, vagina, vas deferens, and all gross internal lesions.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: brain, spleen, and thymus weights for 1 pup/sex/litter. Morphology of developmental and reproductive systems assessed and all lesions retained for examination.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

All analyses conducted using 2-tailed tests, with minimum significance set at 5%.
The following statistical tests were used:
Chi-square, one-way ANOVA with Dunnett’s test, Kruskal-Wallis test with Mann-Whitney U-test, Kolmogorov-Smirnov test (one-tailed), and ANCOVA (with litter size as covariant) and Student’s t-test.

Reproductive indices:

Mating and fertility

Offspring viability indices:

survival

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No significant effects

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Statistically significant differences between the 0 and 2000 ppm exposure groups were observed for body weight gains for weeks 0 to 1 and 1 to 2 (considered to be exposure-related).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No significant effects

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No significant effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No significant effects

ORGAN WEIGHTS (PARENTAL ANIMALS)
Significant increases in mean liver weights (absolute and relative) were observed in the 2000 ppm group males and females. Significant increases in mean absolute and relative kidney weights were observed for males in all exposure groups relative to the control group; however, mean kiney weights of female rats were unaffected.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No significant effects

HISTOPATHOLOGY (PARENTAL ANIMALS)
No significant effects

Key result

Dose descriptor:

NOAEL

Remarks:

parental systemic toxicity

Effect level:

1 000 ppm (analytical)

Based on:

not specified

Sex:

male/female

Basis for effect level:

other: Transient reduced body weight gain and food consumption (systemic toxicity apart from alpha 2µ globulin-mediated nephropathy), increases in liver and kidney weights

Remarks on result:

other: Generation not specified (migrated information)

Key result

Dose descriptor:

NOAEL

Remarks:

neonatal toxicity

Effect level:

1 000 ppm (analytical)

Based on:

not specified

Sex:

male/female

Basis for effect level:

other: Based on observed acute changes on PND 22 and 28 (single mortality and anesthetic CNS effects observed during exposure only).

Remarks on result:

other: Generation not specified (migrated information)

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

2 000 ppm (analytical)

Based on:

not specified

Sex:

male/female

Basis for effect level:

other: No effects observed at highest dose tested

Remarks on result:

other: Generation not specified (migrated information)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
No significant effects.

CLINICAL SIGNS (OFFSPRING)
No significant effects

BODY WEIGHT (OFFSPRING)
No significant effects


ORGAN WEIGHTS (OFFSPRING)
No significant effects

GROSS PATHOLOGY (OFFSPRING)
No significant effects

HISTOPATHOLOGY (OFFSPRING)
No significant effects

OTHER FINDINGS (OFFSPRING)
A single mortality and signs of CNS depression were observed in 18 animals in the 2000 ppm F1 group (7 males and 11 females) following exposure on PND 22 to 25. Due to this observation, exposure in all groups of F1 weanlings was suspended until PND 27. CNS depressive effects were observed in 6 males in the 2000 pm group again on PND 23 to 31, but not again thereafter. Animals in the 1000 and 2000 ppm groups showed reduced reactivity to novel stimulus during exposure.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 000 ppm (analytical)

Based on:

not specified

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: No significant effects.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 ppm (analytical)

Based on:

not specified

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: No significant effects.

Reproductive effects observed:

not specified

Parameter	0 ppm	500 ppm	1000 ppm	2000 ppm
F0
Male mating index	100%	100%	90%	100%
Female mating index	100%	100%	90%	100%
Male fertility index	93.3%	96.7%	86.7%	93.1%
Female fertility index	93.3%	96.7%	86.7%	93.3%
Mean pre-coital interval (days)	2.2 ± 1.21	2.5 ± 1.20	2.7 ± 1.11	3.0 ± 1.66
Gestation length (days)	21.8 ± 0.44	22.2 ± 0.62*	21.7 ± 0.46	22.0 0.19
Testicular sperm numbers (left; million sperm/g tissue)	87.3 ± 9.90	91.9 ± 13.48	91.7 ± 17.26	86.0 ± 15.35
Epididymal sperm numbers (left; million sperm/g tissue)	343.6 ± 92.66	310.1 ± 77.60	355.5 ± 81.06	380.2 ± 92.44
Sperm production rate (million sperm/g tissue/day; left testis)	14.3 ± 1.63	15.1 ± 2.21	15.0 ± 2.83	14.1 ± 2.50
Sperm motility (% motile)	80.0 ± 14.45	78.4 ± 16.38	82.9 ± 9.68	78.3 ± 17.89
Morphologically normal sperm (%)	99.0 ± 1.31	99.0 ± 1.12	99.3 ± 1.42	99.0 ± 1.02
F1
Number born	13.0 ± 2.28	12.5 ± 2.91	13.3 ± 2.22	14.0 ± 2.31
Sex at birth (% male)	52.1 ± 17.53	45.6 ± 19.46	47.9 ± 17.83	54.1 ± 12.99
Live litter size (PND 0)	13.0 ± 2.35	12.4 ± 2.99	13.1 ± 2.18	13.8 ± 2.25
Mean body weight on PND 1 (g)	6.9 ± 0.60	7.1 ± 0.70	6.9 ± 0.50	7.1 ± 0.49
Male mating index	100%	93.3%	93.3%	100%
Female mating index	100%	93.3%	93.3%	100%
Male fertility index	96.7	90.0	80.0	93.3
Female fertility index	96.7	90.0	80.0	93.3
Mean pre-coital interval (days)	3.5 ± 2.73	3.2 ± 2.03	3.5 ± 2.66	3.2 ± 2.28
Gestation length (days)	21.7 ± 0.53	21.7 ± 0.54	21.6 ± 0.58	21.6 ± 0.49
Testicular sperm numbers (left; million sperm/g tissue)	86.6 ± 20.21	89.3 ± 12.24	82.6 ± 18.69	81.3 ± 10.86
Epididymal sperm numbers (left; million sperm/g tissue)	517.5 ± 124.37	507.1 ± 106.48	550.3 ± 141.39	540.9 ± 93.27
Sperm production rate (million sperm/g tissue/day; left testis)	14.2 ± 3.31	14.6 ± 2.01	13.5 ± 3.06	12.9 ± 3.00
Sperm motility (% motile)	80.8 ± 13.29	82.2 ± 10.52	84.4 ± 10.30	83.9 ± 9.94
Morphologically normal sperm (%)	98.7 ± 4.07	99.0 ± 1.21	99.0 ± 0.82	98.9 ± 0.82
F2
Number born	13.4 ± 2.86	13.8 ± 1.96	13.5 ± 2.36	14.2 ± 1.87
Sex at birth (% male)	48.6 ± 14.54	49.9 ± 14.06	45.8 ± 12.02	50.2 ± 10.51
Live litter size (PND 0)	13.1 ± 2.82	13.6 ± 2.19	13.5 ± 2.36	14.0 ± 1.89
Mean body weight on PND 1 (g)	6.9 ± 0.59	6.8 ± 0.65	6.8 ±0 .64	6.6 ± 0.48

Conclusions:

Although reproductive/developmental parameters were not affected by inhalation of MIBK at concentrations up to 2000 ppm, over 2 generations in rats, adverse liver and kidney effects in adult rats were apparent.  THE NOAEL for parental systemic toxicity and neonatal toxicity was considered to be 1000 ppm. The NOAEL for reproductive toxicity was considered to be 2000 ppm.

Executive summary:

The reproductive toxicity of methyl isobutyl ketone (MIBK) was evaluated in a GLP-compliant multi-generation toxicity study in Crj: CD(SD) rats. The study design was equivalent to OECD test guideline 416. Rats were administered MIBK at target concentrations of 0, 500, 1000 and 2000 ppm (mean measured concentrations were 0, 491, 999, and 1996 ppm or 0, 2012, 4093, and 8178 mg/m3) by whole body inhalation. Parental (F0) findings included transient decreased body weight during the first 2 weeks of exposure at the 2000 ppm dose concentration and increases in absolute and relative liver weights at 2000 ppm. Significant increases in parental F0 and F1 mean absolute and relative kidney weights were observed for males in all MIBK-treated groups relative to the control group; however, mean kidney weights of female rats were unaffected. These increases in mean kidney weight were attributed to an alpha2µ-mediated mechanism and are not considered relevant to human risk identification (see Section 7.9.3). Offspring findings included a single mortality and signs of CNS depression in the F1 group following MIBK exposure on postnatal day (PND) 22 to 25. As a result, F1 MIBK exposure was suspended until PND 27. CNS depressive effects were observed until PND 31, but not after. F1 animals in the 1000 and 2000 ppm groups showed reduced reactivity to novel stimulus during exposure, which was attributed to a sedative effect. There were no effects on reproductive parameters reported. Based on these findings the NOAEL for parental systemic toxicity and neonatal toxicity was considered to be 1000 ppm. The NOAEL for reproductive toxicity was considered to be 2000 ppm, the highest dose tested.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

4 093 mg/m³

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The reproductive toxicity of methyl isobutyl ketone (MIBK) was evaluated in a GLP-compliant multi-generation toxicity study in Crj: CD(SD) rats (Nemec, 2000; Nemec et al., 2004). The study design was equivalent to OECD test guideline 416. Rats were administered MIBK at target concentrations of 0, 500, 1000 and 2000 ppm (mean measured concentrations were 0, 491, 999, and 1996 ppm or 0, 2012, 4093, and 8178 mg/m³) by whole body inhalation. Parental (F0) findings included transient decreased body weight during the first 2 weeks of exposure at the 2000 ppm dose concentration and increases in absolute and relative liver weights at 2000 ppm. Significant increases in parental F0 and F1 mean absolute and relative kidney weights were observed for males in all MIBK-treated groups relative to the control group; however, mean kidney weights of female rats were unaffected. These increases in mean kidney weight were attributed to an alpha2µ-mediated mechanism and are not considered relevant to human risk identification. Offspring findings included a single mortality and signs of CNS depression in the F1 group following MIBK exposure on postnatal day (PND) 22 to 25. As a result, F1 MIBK exposure was suspended until PND 27. CNS depressive effects were observed until PND 31, but not after. F1 animals in the 1000 and 2000 ppm groups showed reduced reactivity to novel stimulus during exposure, which was attributed to a sedative effect. There were no effects on reproductive parameters reported. Based on these findings the NOAEL for parental systemic toxicity and neonatal toxicity was considered to be 1000 ppm (4093 mg/m³). The NOAEL for reproductive toxicity was considered to be 2000 ppm (8178 mg/m³), the highest dose tested.

Short description of key information:
A GLP inhalation multi-generation study in rats, equivalent to OECD Guideline 416, reported a parental and neonatal toxicity of 1000 ppm (4093 mg/m3 at 20 °C), and a reproductive toxicity of 2000 ppm (8330 mg/m3 at 20 °C; the highest dose tested). Reproductive tests in other species, or by the dermal or oral dose route, were not conducted.

Justification for selection of Effect on fertility via inhalation route:
Key study

Effects on developmental toxicity

Description of key information

In inhalation developmental toxicity studies in rats and mice, maternal toxicity and fetotoxicity were seen at 3000ppm (12292 mg/m3). Effects in the dams included decreased body weight gain, increased liver and kidney weights, decreased food consumption, and in mice, maternal deaths. Reduced fetal body weights and delayed ossification were noted in both species and increased resorptions were noted for mice. 1000ppm (4106 mg/m3) was considered to be the NOEL for both maternal animals and offspring.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

4 106 mg/m³

Species:

other: rat and mouse

Additional information

Developmental and maternal toxicity were evaluated in groups of 35 pregnant Fischer 344 rats and 30 pregnant CD-1 mice exposed by inhalation to 0, 300, 1000, or 3000 ppm (0, 1229, 4106, 12,292 mg/m³) MIBK for 6 hrs/day on gestation days 6 through 15 (Tyl, 1984; Tyl et al., 1987). Animals were sacrificed on gestation day 21 (rats) or 18 (mice). Dams were evaluated for exposure-related changes in clinical signs, body weight, food consumption, organ weights (kidney, liver, and gravid uterus), and reproductive parameters; fetuses were evaluated for exposure-related changes in body weight and viability, and for external, skeletal, and thoracic and peritoneal visceral alterations. Maternal mean body weight, weight gain, and food consumption were significantly decreased in rats exposed to 12,292 mg/m³(but not to 4106 mg/m3 or lower) during the exposure period, but they had recovered to control levels by the day of sacrifice; maternal body weight was not affected in mice. Maternal clinical signs observed in rats or mice included coordination loss, hindlimb weakness, paresis, irregular gait, hypoactivity, ataxia, unkempt fur, negative tail or toe pinch, piloerection, lacrimation, or red perioral encrustation. These clinical signs were observed only during the exposure period and only at 12,292 mg/m3. Three maternal deaths (12% of the animals in the group) occurred in mice exposed to 12,292 mg/m³after the first exposure on gestation day 6; no further deaths occurred in that group, and no exposure-related deaths occurred in the other mouse or rat exposure groups. Neonates from those dams were not considered in the final evaluation. Statistical analyses by the authors were per dam or per litter. No exposure-related effects were observed in rats or mice with respect to numbers of corpora lutea, total implants, percent implantation loss, live fetuses per litter, nonviable implants per litter, percent live fetuses, and sex ratio. In mice, there was an increased mean number of dead fetuses per litter at 12,292 mg/m³(0.6 per litter compared to 0.1 in controls). Fetal body weights (litter weight, male weight per litter, and female weight per litter) were significantly reduced in rats exposed to 1229 (the mean by 3%) and 12,292 mg/m³(the mean by 6%) but not to 4106 mg/m³; and in mice, fetal body weights were statistically significantly reduced at 12,292 mg/m³(the mean by 13%) but not at 4106 mg/m³or below. The authors indicated that the reduction in rat fetal body weight was confounded by a skewed distribution of litter size, whereby higher doses had very small litters and smaller litters had varied mean weights across dose, while lower-dosed dams appeared to have larger litters and larger litters showed a dose-dependence in mean weight. There was no statistically significant increase in the number of rat or mouse fetuses per litter. The authors decided the reductions in rat fetal body weight was not treatment-related. No exposure-related change in the incidence of malformations of any type were observed in rat and mouse fetuses. The number of litters with observations indicating retarded skeletal ossification was significantly increased to various degrees in both rats and mice at 12,292 mg/m³relative to controls for a variety of skeletal endpoints, with scattered increases in litters with retarded ossification at lower exposure levels that were not considered to be exposure-exposure-related.

Justification for selection of Effect on developmental toxicity: via inhalation route:
Reliable inhalation developmental toxicity studies are available in rats and mice.

Justification for classification or non-classification

The substance does not meet the criteria for classification and labelling for this endpoint, as set out in Regulation (EC) NO. 1272/2008.