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Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to Category Document

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000
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 et al.,
Year:
2004
Bibliographic source:
Int J Toxicol, 23, 127-143

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Methyl isobutyl ketone
- Physical state: liquid
- Analytical purity: study report states that purity is reported in Appendix A (Appendix not available at time of entry)
- Lot/batch No.: Lot-97-J-14S and Lot-98-1-3S
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals 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

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 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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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

Effect levels (P0)

Key result
Dose descriptor:
NOAEC
Remarks:
parental systemic toxicity
Effect level:
1 000 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios

Results: P1 (second parental generation)

Effect levels (P1)

Key result
Dose descriptor:
NOAEC
Effect level:
1 000 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
1 000 ppm (analytical)
Based on:
test mat.
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).

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
2 000 ppm (analytical)

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:
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.