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Toxicological information

Specific investigations: other studies

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Administrative data

Endpoint:
specific investigations: other studies
Remarks:
Lactational transfer
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The study was conducted as part of an OECD 443 extended 1-generation study to demonstrate that the test article or its metabolites can be transfered to the offspring via milk.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
The principle of the study is to conduct an analysis of lactating animals exposed to the test article to determine if the test article can be found.
GLP compliance:
yes
Type of method:
in vivo
Endpoint addressed:
other: Lactational transfer

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
no data
Specific details on test material used for the study:
Identification: Methyl isoamyl ketone (also referred to as MiAK; CAS No. 110 12-3)
Batch/Lot No.: TD16040801
Receipt Date: 05 Sep 2017
Physical Description: Clear, colorless liquid
Purity: 99.33%
Water Content: 0.0084%
Storage Conditions: Kept in a controlled temperature area set to maintain 18°C to 24°C
Supplier: Eastman Chemical Company

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
Housing
On arrival, the F0 animals were group housed (up to 3 animals of the same sex) until cohabitation. During cohabitation, the animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding (Bed O’ Cobs®) equipped with an automatic watering valve.
Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal number(s), and sex. Cages were arranged on the racks in group order.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International.

Environmental Conditions
Target temperatures of 68°F to 78°F (20°C to 26°C) with a relative target humidity of 30% to 70% were maintained. A 12-hour light/12-hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
PMI Nutrition International, LLC Certified Rodent LabDiet® 5K96 Advanced Protocol® Verified Casein Diet 10 IF was provided ad libitum throughout the study, except during designated procedures. The feed used on this study needed to have a genistein-equivalent (aglycone) content of less than 300 ppm. PMI Nutrition International, LLC Certified Rodent LabDiet® 5K96 Advanced Protocol® Verified Casein Diet 10 IF consistently analyzed at less than 10.0 ppm total isoflavones (aglycone equivalents of genistein, daidzein, and glycitein).
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility.
It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system, except during designated procedures.
Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility.
It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Animal Enrichment
Animals were socially housed for psychological/environmental enrichment and were provided with environmental enrichment as appropriate to aid in maintaining the animals’ oral health, except during designated procedures.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
air
Details on exposure:
Filtered air or MiAK vapor was administered as 6-hour, daily whole-body inhalation exposures, 7 days per week, at approximately the same time each day, according to the schedule below.
F0 females were exposed to either filtered air or vaporized MiAK concurrently with, and in the same exposure chambers, as animals on a concurrent extended one-generation reproductive toxicity study in Sprague Dawley rats.1 For all females, exposures were conducted for a minimum of 14 days prior to mating and continuing throughout the mating period. Following completion of the mating period, females (with no evidence of mating) continued to be exposed daily until 1 day prior to scheduled euthanasia. Males were not exposed to the test substance, and were only used for breeding purposes.
For females with evidence of mating, exposures during gestation were conducted up to and including Gestation Day 20, at which time exposure was suspended, through Lactation Day 4, to avoid confounding effects on parturition and nursing behavior. Exposures resumed on Lactation Day 5, and continued until Lactation Day 13, inclusively. During lactation, dams were separated from their litters during the exposure period.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
14 days pre-mating, maximum 2 weeks for mating, followed by gestation and to sacrifice at lactation day 13
Frequency of treatment:
daily
Post exposure period:
none
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
1 500 ppm
Dose / conc.:
750 ppm
Dose / conc.:
380 ppm
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
The study design involved exposing female rats to the test article beginning prior to gestation, throughout pregnancy and into lactation, with an analysis of milk and pup plasma for the test article.

Examinations

Examinations:
Observations
Clinical observations were performed once daily throughout the study. During the exposure period, these observations were performed prior to exposure. On exposure days, observations were also recorded at the approximate mid-point of exposure (animals visible in exposure chamber only), at the end of exposure, and 1–2 hours postexposure (see Appendix 1 - Study Protocol and Deviations).
During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

Body Weights
F0 females were weighed individually weekly. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 10, and 13.

Food Consumption
Food consumption was quantitatively measured weekly throughout the study period. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 10, and 13.
Positive control:
No

Results and discussion

Details on results:
Whole-body inhalation of MiAK to female Crl:CD(SD) rats for 6 hours daily for at least 14 days prior to mating and continuing throughout the mating, gestation, and lactation periods until Lactation Day 13 resulted in quantifiable levels of MiAK in maternal milk and plasma that increased in a slightly greater than dose-proportional manner from 380 ppm to 750 ppm and roughly increased 2x from 750 ppm to 1500 ppm. For all groups, mean MiAK concentrations in milk were approximately 16-fold higher than in plasma. Even though plasma MiAK concentrations in pups were BLQ (<1000 ng/mL) at all exposure concentrations, the maternal plasma and milk concentration data demonstrate that the offspring were exposed to MiAK during both the gestation (in utero) and neonatal periods (through nursing).

Any other information on results incl. tables

Plasma MiAK Concentrations on Lactation/Postnatal Day 13 (ng/mL)

Target Exposure Concentration (ppm) Lactation Day 13 Postnatal Day 13
Dams Pups
0 Mean All BLQ < (1000) All BLQ < (1000)
S.D.
380 Mean 483 All BLQ < (1000)
S.D. 881
750 Mean 1540 All BLQ < (1000)
S.D. 2170
1500 Mean 3100 All BLQ < (1000)
S.D. 5840

Milk MiAK Concentrations on Lactation Day 13 (ng/mL)

Target Exposure Concentration (ppm) LD 13
Females
0 Mean All BLQ<(1000)
S.D.
380 Mean 7780
S.D. 6610
750 Mean 24,400
S.D. 19,200
1500 Mean 51,700
S.D. 55,900

Applicant's summary and conclusion

Conclusions:
Whole-body inhalation of MiAK to female Crl:CD(SD) rats for 6 hours daily for at least 14 days prior to mating and continuing throughout the mating, gestation, and lactation periods until Lactation Day 13 resulted in quantifiable levels of MiAK in maternal milk and plasma that increased in a slightly greater than dose-proportional manner from 380 ppm to 750 ppm and roughly increased 2x from 750 ppm to 1500 ppm. For all groups, mean MiAK concentrations in milk were approximately 16-fold higher than in plasma. Even though plasma MiAK concentrations in pups were BLQ (<1000 ng/mL) at all exposure concentrations, the maternal plasma and milk concentration data demonstrate that the offspring were exposed to MiAK during both the gestation (in utero) and neonatal periods (through nursing).
Executive summary:

The objective of this study was to detect the potential of the test substance, methyl isoamyl ketone (hereafter referred to as MiAK), to be secreted in maternal milk and to assess exposure of the pups via assessment of MiAK in maternal and pup plasma. F0females were exposed via whole-body inhalation for 6 hours daily for at least 14 days prior to mating and continuing throughout the mating, gestation, and lactation periods until Lactation Day 13, with the exception that exposure was suspended from Gestation Day 21 through Lactation Day 4. A concurrent control group was exposed to humidified, filtered air on a comparable regimen. Overall mean analyzed exposure concentrations were 380, 748, and 1500 ppm for Groups 2, 3, and 4, respectively.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, reproductive performance, parturition, litter viability and survival, and exposure assessment in milk and plasma.

All F0females in the control, 380, 750, and 1500 ppm groups survived to the scheduled necropsy. Test substance-related increased incidences of red material around the nose in the 750 and 1500 ppm groups and red material around the mouth in the 1500 ppm group were noted at the end of exposure and 1–2 hours postexposure. In addition, increased incidences of hair loss on various body surfaces were noted for females in the 1500 ppm group at the daily examinations. No other test substance‑related clinical findings were noted at the daily examinations or at the midpoint of exposure, end of exposure, and 1–2 hours postexposure. 

Test substance-related lower mean body weight gains, with corresponding effects on mean food consumption, were generally noted for females in the 1500 ppm group throughout the pre‑mating, gestation, and lactation periods, resulting in mean body weights that were lower (up to 5.9%) than the control group. In addition, a lower mean body weight gain was noted for females in the 750 ppm group during Study Days 7–13, without corresponding effects on mean food consumption or mean body weights. No other effects on mean body weights, body weight gains, and food consumption were noted at any exposure concentration.

No test substance-related effects were noted on reproductive performance (female mating, fertility, and conception), gestation length, or the process of parturition at any exposure concentration.

Survival, clinical condition, and growth of the F1generation was unaffected by test substance exposure at all exposure concentrations.

In conclusion, whole-body inhalation of MiAK to female Crl:CD(SD) rats for 6 hours daily for at least 14 days prior to mating and continuing throughout the mating, gestation, and lactation periods until Lactation Day 13 resulted in quantifiable levels of MiAK in maternal milk and plasma that increased in a slightly greater than dose-proportional manner from 380 ppm to 750 ppm and roughly increased 2x from 750 ppm to 1500 ppm. For all groups, mean MiAK concentrations in milk were approximately 16-fold higher than in plasma. Even though plasma MiAK concentrations in pups were BLQ (<1000 ng/mL) at all exposure concentrations, the maternal plasma and milk concentration data demonstrate that the offspring were exposed to MiAK during both the gestation (in utero) and neonatal periods (through nursing).