Butanone

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

September 24, 1998 to December 2, 1998.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Reference 2

Reference Type:

publication

Title:

Inhalation two-generation reproductive toxicity study of methyl isobutyl ketone in rats.

Author:

Nemec MD, Pitt JA, Topping DC, Gingell R, Pavkov KL, Rauckman EJ, Harris SB

Year:

2004

Bibliographic source:

International journal of toxicology 23: 127-143

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-methylpentan-2-one

EC Number:

203-550-1

EC Name:

4-methylpentan-2-one

Cas Number:

108-10-1

Molecular formula:

C6H12O

IUPAC Name:

4-Methyl-2-pentanone

Test material form:

liquid

Test animals

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: PMI Nutrition International Certified Rodent LabDiet® 5002, ad libitum
- Water: municipal water treated on-site by reverse osmosis, ad libitum
- Acclimation period: 23 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes per hour: 10 during acclimation, 12 to 15 during exposure
- Photoperiod (hrs dark / hrs light): 12/12

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

Administration / exposure

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 applicable
- Temperature, humidity, pressure in air chamber: 22 ± 2°C and 30 to 70%, respectively
- Air flow rate: 12 to 15 air changes per hour
- Air change rate: 12 to 15 per hour
- Method of particle size determination: not applicable
- Treatment of exhaust air: not reported

TEST ATMOSPHERE
- Brief description of analytical method used: exposure concentrations were measured 9 to 10 times during each daily exposure by a validated gas chromatographic method. Samples were also taken from exposure containers before and after use, to check stability. Compound structure was verified using GC-MS.
- 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: presence of copulatory plug or of sperm in vaginal smear, referred to as day 0 of gestation
- After 14 days of unsuccessful pairing, females were placed in a maternity cage with nesting material.
- Further mating after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Exposure concentrations were measured 9 to 10 times (approximately every 35 minutes) during each daily exposure by a validated gas chromatographic method.

Duration of treatment / exposure:

F0 and F1 males: at least 70 days prior to mating until 1 day prior to euthanasia
F0 and F1 females: at least 70 days prior to mating until gestational day 20; then from PND day 5 until 1 day prior to euthanasia
F1 litters were potentially exposed in utero and during PND days 0-21, and were intentionally exposed from weaning PND 22 and continued.
F2 animals were potentially exposed in utero and during lactation day 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Remarks:

Target concentration. Analytical concentrations were 491 ppm (F0) and 506 ppm (F1), equivalent to 2011 mg/m³ and 2073 mg/m³ respectively.

Doses / concentrations 2

Dose / conc.:

1 000 ppm

Remarks:

Target concentration. Analytical concentrations were 999 ppm (F0) and 1002 ppm (F1), equivalent to 4092 mg/m³ and 4105 mg/m³ respectively.

Doses / concentrations 3

Dose / conc.:

2 000 ppm

Remarks:

Target concentration. Analytical concentrations were 1996 ppm (F0) and 2006 ppm (F1), equivalent to 8177 mg/m³ and 8217 mg/m³ respectively

No. of animals per sex per dose:

30

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: on the basis of previous studies with test article
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: not applicable

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: appearance, behaviour, pharmacotoxic signs within one hour after completion of exposure, moribundity and mortality. In addition, at the approximate midpoint of each day’s exposure, a Striker device was used to produce a loud noise (novel stimulus) directly on the front glass of the exposure chamber. The animals in accessible cages were observed each day for a reaction to the stimulus, and the findings were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

Oestrous cyclicity (parental animals):

The oestrous stage of each female F0 and F1 was determined by daily preparation of vaginal smears beginning 21 days before pairing and continuing until evidence of mating was present or the end of the mating period. The oestrous 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 (oesophagus, 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. The morphology of developmental and reproductive systems was 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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the mid dose group died during parturition; clinical findings for two days before death indicated dystocia. One male was euthanised in extremis due to mechanical injury to nose. Neither death was attributed to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Transient reductions in mean body weight gains were observed in the high dose group F0 females during weeks 0-1 and 1-2. No effects were observed in F1 females at these intervals. Mean body weights were unaffected in F0 mid and low dose groups, and in all F0 males at all exposure levels.

Food efficiency:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There was an increase in the number of observations of male and female rats having no reaction to an auditory startle stimulus in the mid and high dose groups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No significant effects were observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Individual variation in the oestrous cycle occurred in all study groups. The regularity and duration of oestrus were not affected by exposure.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

Transient reduced body weight gain and food consumption were observed in high dose males and females. Sedative effects were observed in the high dose group (lower response to auditory stimulus during exposure); these effects were not evident 1 hour after exposure. No reproductive toxicity was observed at any dose level.

Effect levels (P0)

1

Dose descriptor:

NOAEC

Remarks:

parental systemic toxicity

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEC

Remarks:

reproductive toxicity

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed at any dose level.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Exposure-related clinical signs, including rocking, lurching or swaying while walking, half-closed eyelids, bilateral lacrimation and red material around the nose and mouth, were observed in the 2000 ppm group F1 (P1) males and females. As a result of this mortality and the clinical signs observed, exposure was suspended to PND 27. After exposure was resumed, these clinical signs were observed in 6 out of 30 high dose group males, but not in females. The predominant clinical sign observed for F1 parental (P1) males consisted of hair loss on the forelimbs.

Mortality:

mortality observed, treatment-related

Description (incidence):

One F1 (P1) male in the high dose group died after one day of exposure, on PND 22, and was replaced by another weanling. No further mortality occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight gains were reduced in the 2000 ppm group F1 parental (P1) males and females during week 17-18 (the first week of exposure following selection) and in the 2000 ppm group F1 parental (P1) males during week 18-19. 19. Mean body weights in the 2000 ppm group F1 parental (P1) females were slightly reduced throughout the pre-breeding and post-lactational phases. These transient reductions, although slight, were attributed to test article exposure. Mean body weights and body weight gains were unaffected by test article exposure throughout gestation and lactation for F1 parental (P1) females at all exposure levels evaluated.

Food efficiency:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

A dose-related increase in the numbers of animals of both sexes with no response to an auditory startle stimulus was observed in the mid and high dose groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Exposure-related increases in mean absolute and relative (to final body weight) liver weights were observed for F1 parental (P1) males and females at the 2000 ppm level; relative liver weights were also increased for F1 parental (P1) males at the 1000 ppm level. Correlating exposure-related centrilobular hepatocellular hypertrophy was observed - this is considered an adaptive response and not indicative of systemic toxicity.
Exposure-related increases in mean absolute and relative (to final body weight) kidney weights were observed for F1 parental (P1) males and females in all exposure groups. Microscopic changes were observed in basophilic tubules for all dose groups, with corresponding acidophilic hyaline inclusions consistent with alpha 2µ globulin formation observed at all exposure levels. This is known to be a male rat specific effect.

Gross pathological findings:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P1)

Transient reduced body weight gain and food consumption were observed in high dose males and females. Sedative effects were observed in the high dose group (lower response to auditory stimulus during exposure); these effects were not evident 1 hour after exposure. No reproductive toxicity was observed at any dose level.

Effect levels (P1)

1

Dose descriptor:

NOAEC

Remarks:

F1 parental (P1) systemic toxicity

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

2

Dose descriptor:

NOAEC

Remarks:

reproductive toxicity

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed at any dose level

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Details on results (F1)

Mean pup body weights, sex ratios, live litter sizes, numbers of dead pups on lactation day 0 and viability indices were unaffected by exposure to the test article. Various indicators of physical development, as well as behavioural responses, of the FI pups were comparable to the control group values. No test article-related internal findings were noted in the F1 pups that died or were euthanised, or at the scheduled necropsies. Necropsy findings, organ weights and microscopic findings for the selected F1 weanling pups did not suggest any effects of exposure to the test article. There were no effects on live litter sizes, number of dead pups and viability.

Effect levels (F1)

Dose descriptor:

NOAEC

Remarks:

neonatal toxicity

Generation:

F1

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect observed in F1 pups which were not subsequently exposed to the test material

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

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

Details on results (F2)

Mean pup body weights, sex ratios, live litter sizes, numbers of dead pups on lactation day 0 and viability indices were unaffected by exposure to the test article. There were no effects on live litter sizes, number of dead pups and viability.

Effect levels (F2)

Dose descriptor:

NOAEC

Remarks:

neonatal toxicity

Generation:

F2

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

Methyl isobutyl ketone (MIBK) has been tested in a whole-body inhalation study. Male and female Sprague-Dawley rats were exposed to 0, 500, 1000 or 2000 ppm MIBK, 6 hours/day, 7 days/week for 70 days premating and from mating through gestation day 20 and from postnatal day 5 for F0 and F1 females. The NOAEC for parental systemic toxicity and neonatal toxicity was considered to be 1000 ppm. The NOAEC for reproductive toxicity was considered to be 2000 ppm, the highest dose tested.

Executive summary:

The reproductive toxicity of methyl isobutyl ketone (MIBK) has tested in a two-generation toxicity study in Crj: CD(SD) rats conducted according to a protocol similar to OECD TG 416 and in compliance with GLP. Rats were administered MIBK at target concentrations of 0, 500, 1000 and 2000 ppm by whole body inhalation. Mean measured concentrations for the F0 generation were 0, 491, 999, and 1996 ppm, equivalent to 0, 2011, 4092, and 8177 mg/m³; for the F1 generation mean measured concentrations were 0, 506, 1002 and 2006, equivalent to 0, 2073, 4105 and 8217mg/m³. Parental (F0 and F1) 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 parental 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 parental 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 NOAEC for parental systemic toxicity and neonatal toxicity was considered to be 1000 ppm. The NOAEC for reproductive toxicity was considered to be 2000 ppm, the highest dose tested.