Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Remarks:
developmental endpoints investigated as part of a two generation reproductive toxicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 24 OCT 2005 to 25 JUN 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study but not specifically relating to developmental toxicity
Cross-reference
Reason / purpose for cross-reference:
other: read-across target

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010
Reference Type:
publication
Title:
Unnamed
Year:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-butyl acetate
EC Number:
204-658-1
EC Name:
N-butyl acetate
Cas Number:
123-86-4
Molecular formula:
C6H12O2
IUPAC Name:
butyl acetate
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): n-butyl acetate
- Stability under test conditions: confirmed analytically to be stable under storage conditions
- Storage condition of test material: refrigerated
- Analytical purity: 99.8%
- Impurities (identity and concentrations): Butanol: 0.1%
- Purity test date: 2005-06-06
- Lot/batch No.: Lot# 54061404312985 Batch# TE1601FTC8
- Expiration date of the lot/batch: 2007-06-02

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS (Crl:CD (SD))
- Source: Charles River Laboratories, Inc. Raleigh, North Carolina, USA
- Age at study initiation: (P) 7 wks
- Weight at study initiation: (P) Males: 207 g to 263 g ( group means: 235-237 g); Females: 151 to 201 g (group means: 169-171 g)
- Fasting period before study: no
- Housing: 3 per cage (by sex) for the initial acclimation period; individually thereafter except for mating period and after parturition; following weaning dams were individually housed until scheduled necropsy; weaned pups were housed by litter in plastic cages for 1 week, beginning on PND 28, the F1 and F2 pups were inidvidually housed until the start of the mating period (F1) or until euthanasia (F2)
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet 5002; ad libitum except during exposure periods
- Water: reverse osmosis-purified drinking water; ad libitum except during exposure periods
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

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 cubic metre stainless steel and glass whole body exposure chambers/group
- Method of holding animals in test chamber: individually housed in stainless steel wire-mesh caging suspended over cageboard during exposure
- Source and rate of air: provided from a HEPA- and charcoal-filtered, temperature and humidity controlled source
- Method of conditioning air: test article vapour was directed to the exposure chamber inlet where the vapour concentration was reduced to the desired level by mixing with the chamber ventilation air
- System of generating vapour: glass-bead column-type vaporization system
- Temperature, humidity, pressure in air chamber: daily mean chamber temperature: 21 to 22°C; daily mean chamber humidity: 43 to 56%; slight negative pressure
- Air flow rate: mean values: 430 to 475 litters per minute
- Air change rate: at least 12 to 15 per hour
- Treatment of exhaust air: charcoal and HEPA filtration


TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatograph (GC)
- Samples taken from breathing zone: yes; approximately every 35 minute
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- gas chromatography
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility for an additional 7 days.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: plastic maternity cage with nesting material
Duration of treatment / exposure:
- 6 hours per day for at least 70 consecutive days prior to mating
- F0 and F1 females continued to be exposed throughout mating and gestation through gestation day 20; inhalation exposure was suspended from gestation day 21 through lactation day 4
- on lactation days 1 to 4 F0 and F1 maternal animals were exposed via oral gavage
- inhalation exposure of F0 and F1 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia
- inhalation exposure of the F1 was initiated on PND 22
Frequency of treatment:
7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Remarks:
(inhalation exposure)
Dose / conc.:
750 ppm (nominal)
Remarks:
(inhalation exposure)
Dose / conc.:
1 500 ppm (nominal)
Remarks:
(inhalation exposure)
Dose / conc.:
2 000 ppm (nominal)
Remarks:
(inhalation exposure)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
(actual ingested) F0 and F1 maternal animals during lactation day 1-4; administered as 3 equal doses, approximately 2 hours apart
Dose / conc.:
1 125 mg/kg bw/day (nominal)
Remarks:
(actual ingested) F0 and F1 maternal animals during lactation day 1-4; administered as 3 equal doses, approximately 2 hours apart
Dose / conc.:
2 250 mg/kg bw/day (nominal)
Remarks:
(actual ingested) F0 and F1 maternal animals during lactation day 1-4; administered as 3 equal doses, approximately 2 hours apart
Dose / conc.:
3 000 mg/kg bw/day (nominal)
Remarks:
(actual ingested) F0 and F1 maternal animals during lactation day 1-4; administered as 3 equal doses, approximately 2 hours apart
No. of animals per sex per dose:
30/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on range finding study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 and F1 males: weekly
F0 and F1 females: weekly until evidence of copulation; on gestation day 0, 4, 7, 11, 14, 20 and on lactation day 1, 4, 5 (pre-exposure), 7, 14, 21; weekly after weaning until scheduled necropsy
F2 males and females: weekly beginning on PND 28 through euthanasia

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations:
F0 and F1 males: weekly (not recorded during mating period)
F0 and F1 females: weekly until evidence of copulation (not recorded during mating period); on gestation day 0, 4, 7, 11, 14, 20 and on lactation day 1, 4, 5 (pre-exposure), 7, 14, 21; weekly after weaning until scheduled necropsy
F2 males and females: weekly beginning on PND 28 through euthanasia
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OESTROUS CYCLICITY
Vaginal lavages were performed daily and the slides were evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female for 21 days prior to pairing and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P], beginning 21 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of estrus.

SACRIFICE
- Maternal animals: All surviving F0 and F1 animals that delivered were euthanized between 6 and 10 days after wening of their litters. F0 and F1 females that mated but did not give birth were euthanized on presumed gestation day 25. F0 and F1 females that experienced total litter loss were euthanized within 24 hours. F0 and F1 females that failed to mate within the first 14 days of the breeding period were euthanized on gestation day 15 (females that mated with the second male) or post-cohabitation day 15 (females that did not mate with the second male).
Animals underwent complete necropsy and selective histopathologic examination.

GROSS NECROPSY (F0, F1 and F2 adults)
- Gross necropsy consisted of examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities, including viscera.

HISTOPATHOLOGY (F0 and F1 parental animals)
Microscopic evaluations were performed on the following tissues for all F0 and F1 parental animals from the control and high-exposure groups:

Adrenals Vagina
Coagulating glands
Kidneys Spleen
Liver Gross lesions
Lungs Thyroid
Nasal passages (additionally investigated in animals of the 750 ppm group) Uterus
Ovaries
Oviducts
Pituitary

In addition, microscopic evaluations were performed on the following tissues for F0 and F1 parental animals from the low- and mid-exposure groups that did not mate or produce offspring.
Ovaries, Oviducts, Uterus, Pituitary

ORGAN WEIGHTS (F0 and F1 adults)
Adrenals, Brain, Kidneys, Spleen, Liver , Lungs (prior to inflation with fixative), Thyroid, Ovaries, Uterus (with oviducts and cervix), Pituitary
Ovaries and uterine content:
For females that delivered or had macroscopic evidence of implantation, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Numbers of corpora lutea were also recorded for females euthanized on gestation day 15. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.
Fetal examinations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

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

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

SACRIFICE
All surviving F1 and F2 pups not selected for test article exposure were euthanized on PND 21 by carbon dioxide inhalation. F1 and F2 weanlings exposed to the test article beginning on PND 22 but were not selected to continue exposure into the respective maturation phases were euthanized by carbon dioxide inhalation followed by exsanguination between PND 36-47 (F1 generation) and on PND 36 (F2 generation).
Animals underwent necropsy and/or organ weight determination.

- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY (F1 and F2 pups/weanlings)
Gross necropsies with emphasis on developmental morphology and organs of the reproductive system were performed on nonselected F1 and F2 pups euthanized on PND 21, on the F1 weanlings from dam no. 99179 in the 1500 ppm group that were euthanized on PND 28, and on the extra weanlings selected for inhalation exposure beginning on PND 22 that were euthanized between PND 36-47 (F1 generation) and PND 36 (F2 generation).
The following F1 and F2 organs were collected from 3 pups/sex/litter that survived to their
scheduled termination on PND 21. Only gross lesions were collected and preserved for
the F1 and F2 weanlings exposed up to PND 47 or PND 36.

All organs were placed in 10% neutral-buffered formalin (except as noted):

Adrenal glands (2) Seminal vesicles (2)
Brain Spleen
Coagulating glands (2) Testes and epididymides a (2)
Kidneys (2) Thymus
Liver Uterus with cervix
Ovaries (2) Vagina
Pituitary Gross lesions
Prostate

a = If retained, testis and epididymis were fixed in Bouin’ s solution.
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for a minimum significance level of 5%, comparing each test article-exposed group to the control group by sex. Where applicable, the litter was used as the experimental unit.
Parental mating, copulation, conception and fertility indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Mean parental body weights (premating, gestation and lactation) and body weight gains, mean parental food consumption and food efficiency (at each interval), implantation sites, number of unaccounted-for sites, pre-coital intervals, estrous cyclicity, follicular counts (ovary), gestation length, mean day of attainment of pre-weaning and post-weaning developmental landmarks, mean weight on the day of attainment of post-weaning developmental landmarks, mean pup body weights (postnatal period), mean number of pups born, live litter size, organ weights (absolute, relative to body and relative to brain) were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences (Snedecor and Cochran, 1980). If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the control group.
Mean litter proportions (percent per litter) of postnatal pup viability, pup sex ratio at birth, percentage of motile and progressively motile sperm and percentages of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test article-treated groups to the control group. Epididymal and testicular sperm numbers and sperm production rates were analyzed by a t-test (Steel and Torrie, 1980).
Indices:
Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Females Confirmed Pregnant) / Total No. of Males (Females) Used for Mating x 100

Male Fertility Index (%) = No. of Males Siring a Litter / Total No. of Males Used for Mating x 100

Male Copulation Indes (%) = No. of Males Siring a Litter / No. of Males with Evidence of Mating (or Females Confirmed Pregnant) x 100

Female Fertility Index (%) = No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating x 100

Female Conception Index (%) = No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or Females Confirmed Pregnant) x 100

Mean Live Litter Size = Total Viable Pups on PND 0 / No. Litters with Viable Pups on PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection) (% Per Litter) = Summ (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter)/ No. of Litters Per Group x 100

Postnatal Survival for All Other Intervals (% Per Litter) = Summ (Viable Pups Per Litter at End of Interval N/Viable Pups per Litter at Start of Interval N) / No. of Litters Per Group x 100

N = PND 0-1, 1-4 (Pre Selection, 4 (Post Selection)-7, 7-14, 14-21 or 4 (Post Selection)-21

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
P0 Generation
No test article-related clinical findings were noted for animals that died or that survived to the scheduled necropsies.

P1 Generation
No test article-related clinical findings were noted for animals that died or that survived to the scheduled necropsy.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
P0 Generation
- One P0 female in the 2000 ppm group was found dead approximately 3 hours after the first oral gavage dose on lactation day 3. Based on the macroscopic and microscopic examinations, the mortalities in the control group and the lack of mortalities in the 2000 ppm male group, this single death was not considered test article-related.
- One female in the control group WAS euthanized in extremis on study day 37.
- One P0 female in the 750 ppm group experienced a total litter loss on PND 7; this was not considered related to test article exposure in the absence on an exposure-response.
- One P0 female each in the 750 ppm and 2000 ppm groups failed to deliver and were necropsied on post-mating day 25. In addition, 2 females each in the control and 750 ppm groups and 1 female in the 1500 ppm group were necropsied on gestation day or post-cohabitation day 15. All remaining P0 parental animals survived to the scheduled necropsy.

P1 Generation
- One female in the control group, 1 female in the 1500 ppm group and 2 females in the 2000 ppm group were found dead between PND 24 and PND 28. In addition, 2 females in the 1500 ppm group were found dead on lactation day 3 or 4. None of these deaths were considered test article-related because all P1 animals found dead during the week following weaning, weighed at least 19.3% less than their exposed littermate of the same sex and at least 14.3% less than the mean value for their same sex littermates on PND 21 and the deaths during lactation did not occur in an exposure-related manner.
- Five, 4, 0 and 1 females in the control, 750 ppm, 1500 ppm and 2000 ppm group, respectively, failed to deliver and were necropsied on post-mating day 25; an additional female in the 1500 ppm group was euthanized on presumed post-mating day 25. Two and 1 females in the 750 ppm and 1500 ppm groups, respectively, were necropsied on post-cohabitation day or gestation day 15. All remaining P1 parental animals survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
P0 Generation
- Mean body weight gains in the 1500 ppm and 2000 ppm P0 female groups resulted in lower mean cumulative body weight gains (86 g and 79 g, respectively, compared to 106 g in the control group) when the entire pre-mating period (study weeks 0-10) was evaluated. In addition, mean P0 female body weights were 4.4% to 7.4% and 5.3% to 9.3% lower (statistically significant) in these same respective groups when compared to the control group beginning on study weeks 6 and 5, respectively, and continuing throughout the remainder of the pre-mating period
- A slight decreasing trend in mean body weight gain was noted in the 750 ppm P0 male and female groups when compared to the control group. Although statistical significance for lower mean body weight gains was only achieved during study weeks 0-1 and 7-8 for females (none in a manner that was exposure-dependent), the mean cumulative body weight gains for the females (93 g) in this group were lower (statistically significant for females) than the control group (106 g) during the pre-mating period (study weeks 0-10). The slightly lower mean body weight gains noted were not of sufficient magnitude to affect mean body weights when evaluated during the pre-mating exposure periods.
- Mean body weight gains in the 1500 ppm and 2000 ppm groups were generally similar to the control group values during gestation days 0-4, 4-7, 7-11 and 11-14; no statistically significant differences were noted. However, due to statistically significantly lower mean body weight gains noted for both groups during gestation days 14-20, mean body weight gains in these groups were lower than the control group when the entire gestation exposure period (gestation days 0-20) was evaluated; the difference was statistically significant at 2000 ppm.
- Mean P0 maternal body weights in the 1500 ppm and 2000 ppmgroups were statistically significantly lower (5.4% to 8.4% and 9.1% to 11.3%, respectively) throughout lactation when compared to the control group. However, mean body weight gains in these groups were generally similar (and not statistically significant) to the control group when the overall lactation period was evaluated. Therefore, the decreased mean body weights at 1500 ppm and 2000 ppm were considered a continuation of the decreased mean body weights noted during the pre-mating and gestation periods.

P1 Generation
- F1 females in the 1500 ppm and 2000 ppm group had reduced body weight gain during the pre-mating period. During the entire pre-mating period (study weeks 18-29) mean cumulative body weight gains in the 1500 ppm (163 g) and 2000 ppm (159 g) groups were statistically significantly lower than the control group (183 g).
- Lower (statistically significant) mean P1 maternal body weight gains were noted in the 2000 ppm group throughout the gestation period, with the exception of gestation days 11-14, when mean body weight gain was similar (and not statistically different from) the control group. Mean body weights in this group were 13.1% to 16.9% lower than the control group during gestation days 0 to 20; differences from the control group were statistically significant. Mean body weights in the 1500 ppm group were also 8.5% to 10.8% lower (statistically significant) than the control group throughout gestation, with lower (occasionally statistically significant) mean body weight gains during gestation days 0-11 and 14-20. In addition, mean cumulative body weight gains during gestation days 0-20 in the 1500 ppm and 2000 ppm groups (112 g and 100 g, respectively) were statistically significantly lower than the control group value (133 g). Mean body weight gains and body weights of the 750 ppm P1 females were not affected.
- Mean P1 maternal body weights were statistically significantly lower in the 1500 ppm and 2000 ppm groups throughout lactation (5.7% to 8.1% and 12.0% to 14.1%, respectively) when compared to the control group. However, mean body weight gains in these groups were generally similar to the control group when the overall lactation period was evaluated. The only statistically significant differences were lower mean body weight gains noted for both groups during lactation days 7-14. The decreased mean body weights at 1500 ppm and 2000 ppm were considered a continuation of the decreased mean body weights noted during the pre-mating and/or gestation periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
P0 Generation
- Mean weekly food consumption in the 1500 ppm and 2000 ppm P0 female groups was lower than the control group throughout the pre-mating period; the differences were generally statistically significant. Mean weekly food consumption in the 750 ppm P0 female group was slightly lower than the control group during study week 0-1; the g/kg/day difference was statistically significant, and this reduction corresponded to the effect noted on body weight gain during this interval. With the exception of lower food consumption noted during study weeks 7-8 and 8-9 (generally statistically significant), mean food consumption for the females in this group was generally similar to the control group for the remainder of the pre-mating period. The lower food consumption noted for the 750 ppm female group corresponded to the slightly lower body weight gain noted when the entire pre-mating period was evaluated.
- Mean P0 maternal food consumption in the 1500 and 2000 ppm groups were statistically significantly lower than the control group throughout the gestation period. However, there was no corresponding effect on food efficiency at either exposure level.
- During lactation mean food consumption in the 2000 ppm group was lower than the control group, there was no corresponding effect on food efficiency.

P1 Generation
- Mean weekly food consumption (evaluated as g/animal/day) in the 1500 ppm and 2000 ppm P1 female groups was generally lower than the control group during the pre-mating period (study weeks 18-19 through 28-29). Differences from the control group were generally statistically significant. Statistically significant differences in g/kg/day food consumption were noted at 1500 ppm during study week 27-28 (lower than control group and did not occur in an exposure-dependent manner) and at 2000 ppm during study weeks 20-2 1 (higher than control group due to the lower body weights). Mean food consumption and food efficiency in the 750 ppm P1 female group were unaffected by test article exposure.
- Mean P1 maternal food consumption (evaluated as g/animal/day) in the 1500 ppm and 2000 ppm groups was lower (generally statistically significant) than the control group throughout the gestation period. Mean food consumption and food efficiency in the 750 ppm group were unaffected by test article exposure during gestation.
- Mean P1 maternal food consumption (evaluated as g/animal/day) at 750 ppm, 1500 ppm and 2000 ppm was generally similar to the control group during the first week of lactation. During lactation days 7-14 and 14-2 1, mean food consumption in these groups was lower (generally statistically significant) than the control group. However, there was no corresponding effect on g/kg/day food or food efficiency at any exposure level. When the entire lactation period (days 1-21) was evaluated, mean food consumption at all exposure levels was lower than the control group.
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
Description (incidence and severity):
P0 Generation
- Mean relative (to body weight) lung weight was higher as compared to the control values in test article-exposed P0 females - relative to body weight: 0.7%, 5.8%, 8.9%, respectively at 750, 1500 and 2000 ppm ). Only the effects in females at 2000 ppm were statistically significant. Absolute lung weights were generally comparable to controls throughout test article-exposed groups in the presence of lower final body weights. As there were no histologic correlates these results wer not considered adverse.
- Mean adrenal gland weights were higher in the 1500 and 2000 ppm exposure group P0 females as compared to control group values (absolute: 13.0% and 19.6%, respectively; relative to body weight: 29.2% and 37.5%, respectively; relative to brain weight: 13.9% and 21.6%, respectively); this effect was regarded to be stress related.
- Further organ weight changes for which a statistically significant weight change was observed (e.g. effects on brain, spleen, thyroid gland weight ) were considered by the author of the study report to be secondary to changes in body weight.

P1 Generation
- Lung weights relative to body weight were increased in P1 females(2.6%, 3.3%, 5.1%, respectively at 750, 1500 and 2000 ppm), but decreased relative to brain weight. There were no histologic correlates, therefore the effects on lung weight were not considered to be adverse.
- Further organ weight changes for which a statistically significant weight change was observed (e.g. thyroid) were considered by the author of the study report to be secondary to changes in body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test article-related gross necropsy observations in the P0 or the P1 generation.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Test article-related histologic observations were restricted to the nasal cavity and were observed in P0 and P1 females at the 750 and 2000 ppm exposure levels; tissues from the 1500 ppm exposure group were not examined histologically. In the high-exposure (2000 ppm level) animals, degeneration of the olfactory epithelium was most severe and widely distributed at level II, with the dorsal meatus generally affected diffusely and bilaterally, and with multifocal involvement of the nasoturbinates, typically at the tips. Histologic findings were similar at the 750 ppm exposure level, although lesions were less severe and less widely distributed. In both the 750 and 2000 ppm exposure groups, lesions occurred on a gradient, i.e. decreased in incidence, severity and distribution moving caudally within the nasal cavity.
- There were no other test article-related histologic changes in the P0 generation. Remaining histologic findings were considered to be incidental, manifestations of spontaneous diseases or related to some aspect of experimental manipulation other than exposure to the test article. There was no test article-related alteration in the incidence, severity or histologic character of these incidental and spontaneous tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No test article-related effects on P0 or P1 reproductive performance were observed at any exposure level. No test article-related effects on P0 or P1 mean gestation length or the process of parturition were observed at any exposure level.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Remarks:
systemic toxicity, adult rats
Effect level:
750 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: Decrements in mean body weights, body weight gains and/or food consumptions at 1500 and 2000 ppm
Dose descriptor:
LOAEC
Remarks:
local effects, adult rats
Effect level:
750 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: degeneration of the olfactory epithelium in the nasal cavity at 750 and 2000 ppm in males and females (1500 ppm group not investigated)

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 Generation
- Mean F1 male and female pup body weights in the 2000 ppm group were 7.0% and 7.5% lower, respectively, than the control group values on PND 1; the difference for the females was statistically significant. Mean body weight gains in the male and female pups in the 2000 ppm group were generally lower (occasionally statistically significant) compared to the control group throughout the pre-weaning period. As a result, mean F1 male and female pup body weights in this group were 7.9% to 15.1% and 9.3% to 17.5% lower, respectively, than the control group values from PND 4-21; the differences were generally statistically significant.
- Mean F1 male and female pup body weight gains in the 1500 ppm group were lower than the control group values during PND 1-4, 7-14 (statistically significant) and 14-21 and similar to the control group during PND 4-7. As a result, mean F1 male and female pup body weights in the 1500 ppm groups were 5.1% to 11.2% and 6.2% to 12.2% lower, respectively than the control group values during PND 4-21; statistical significance was achieved on PND 14 and 21.
- Mean F1 male and female pup body weight gains and body weights in the 750 ppm group were similar to (and not statistically significantly different from) the control group during the first week of the postnatal period and during PND 14-21. During PND 7-14, mean F1 pup body weight gains in these males and females were slightly but statistically significantly lower than the control group values. These lower mean body weight gains were not of sufficient magnitude to affect mean body weights. Differences in mean pup body weights in the 750 ppm group were slight and not statistically significant compared to the control group.

F2 Generation
F2 litter:
- Mean F2 male and female body weights in the 2000 ppm group were 5.5% and 4.3% lower, respectively, than the control group on PND 1. Mean body weight gains in the 2000 ppm F2 male and female groups were similar to the control group during PND 1-4 (period of oral gavage administration to the F1 maternal animals), but test article-related lower mean body weight gains were noted for both sexes throughout the remainder of the pre-weaning period (PND 4 to 21). The differences from the control group were statistically significant during PND 4-7, PND 7-14, and PND 14-21 for the body weight gains and from PND7 through 21 for body weights. Mean F2 male and female body weights in this group were similar to the control group on PND 4, but were also 8.1% to 14.6% and 9.2% to 15.7% lower, respectively, than the control group during PND 7-21.
- In the 1500 ppm F2 male and female groups, mean pre-weaning body weights and body weight gains were generally similar to the control group during the first week of the pre-weaning period. However, during PND 4-14, mean body weight gains for both sexes in this group were lower than the control group. Statistical significance was achieved during PND 4-7 (females) and 7-14 (both sexes). Mean male and female body weights in the 1500 ppm group were also up to 6.8% and 7.8% lower, respectively, than the control group during PND 14 through 21; the differences were statistically significant for females on both days. Mean male and female F2 pup body weight gains during PND 14-21 were similar to the control group.
- Mean F2 male and female pup body weights and body weight gains in the 750 ppm group were unaffected by test article exposure. No statistically significant differences from the control group were noted.

F2 adults:
- Mean F2 male and female body weight gains in the 2000 ppm group were lower than the control group during the post-weaning direct exposure period. The differences in the males were generally statistically significant and observed throughout the exposure period (PND 21-28 through 49-56), while the statistically significant differences in the females were observed immediately following direct exposure to the test article (PND 2 1-28 and 28-35). As a result, statistically significantly lower mean cumulative body weight gains were noted for the 2000 ppm F2 males (274 g compared to 312 g in the control group during PND 21-63) and females (172 g compared to 191 g in the control group during PND 21-63) compared to the control group. In addition, mean male and female body weights in this group were 12.4% to 15.4% and 10.8% to 17.1% lower, respectively, (statistically significant) than the control group through PND 63.
- Mean body weight gains in the 1500 ppm F2 female group were statistically significantly lower than the control group values during PND 35-42 and 42-49. These transient differences were not observed in an exposure-dependent manner, but were of sufficient magnitude to cause mean cumulative body weight gain in the 1500 ppm F2 females (174 g) to be statistically significantly lower than the control group value (191 g). Although there were no statistically significant differences in mean body weight gains for the F2 males at 1500 ppm during the exposure period, the mean cumulative body weight gain in this group (297 g) was slightly lower (but not statistically significantly different) than the control group (312 g). Mean body weights in the F2 males and females in the 1500 ppm group were up to 6.4% and 9.1% lower, respectively, than the control group values during PND 21-63; the differences in the females were statistically significant during PND 3 5-63.
- Mean body weights, body weight gains and cumulative body weight gains in the 750 ppm F2 male and female groups were generally similar compared to the control group during the entire post-weaning direct exposure period. Differences from the control group were slight and/or not observed in an exposure-dependent manner. Mean F2 male and female body weights in the 750 ppm group were up to 7.7% and 9.8% lower, respectively, than the control group values during PND 2 1-35; the difference on PND 21 was statistically significant for the females. These differences in mean body weights in the 750 ppm F2 male and female groups were attributed to slightly lower mean body weight gains in these groups prior to weaning and direct exposure. Mean body weights in these animals were similar to the control group values for the remainder of the direct exposure period.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
F1 Generation
The mean number of pups born, live litter size, percentage of males per litter at birth and postnatal survival were unaffected by the test article at all exposure levels. Differences from the control group were slight, were not statistically significant and/or did not occur in an exposure-dependent manner.

F2 Generation
F2 litter:
- The mean number of pups born, live litter size, percentage of males per litter at birth and postnatal survival were unaffected by the test article at all exposure levels. Differences from the control group were slight, were not statistically significant and/or did not occur in an exposure-dependent manner.
- The numbers of F2 pups found dead and/or missing, as well as the general physical condition of all F2 pups in this study were unaffected by F1 parental test article exposure. Pups (litters) that were found dead numbered 17(9), 29(12), 5(5) and 15(7) in the control, 750, 1500 and 2000 ppm groups, respectively. Two (2), 6(5), 1(1) and 0(0) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. Sixteen and 10 pups in the litters of female nos. 99137-11 and 99196-12, respectively, in the 1500 ppm group were euthanized and necropsied due to the death of their dam on PND 3 and 4, respectively.

F2 adults:
- One male and one female in the 2000 ppm group were found dead on PND 25 and 22, respectively. Two males and one female in the 750 ppm group were found dead between PND 23 and 31. There were no test article-related clinical findings noted prior to the death of these animals, and based on macroscopic examination, a cause of death could not be determined for these animals. All remaining F2 animals in the control, 750, 1500 and 2000 ppm groups survived to the scheduled necropsy.
Description (incidence and severity):
F1 Generation
The live litter size was unaffected by the test article at all exposure levels.

F2 Generation
F2 litter:
The live litter size was unaffected by the test article at all exposure levels.
Description (incidence and severity):
F1 Generation
- balanopreputial separation: mean age of attainment of the F1 males in the 2000 ppm group was slightly higher than the concurrent control groups (mean age at attainment (PND): 45.6, 47.8*., 46.6 and 47.2 at 0, 750, 1500 and 2000 ppm, respectively); values were within the range of the historical controls (PND 44.2 - 49.0) and did not follow a clear dose response; mean weights at attainment were lower compared to the control group (221.8, 219.8, 200.*1, 195.8* g at 0, 750, 1500 and 2000 ppm); these effects were attributed to the lower body weights noted during the pre-and post-weaning periods.
- vaginal patency: not affected
- No internal findings that could be attributed to parental exposure to the test article were noted at the necropsies of pups that were found dead or pups weaned at PND 21.
- Pinnal detachment: not affected
- Incisor eruption: not affected
- eye opening: not affected

F2 Generation
- balanopreputial separation: mean age of attainment of the F2 males in the 2000 ppm group was slightly higher than the concurrent control groups (mean age at attainment (PND): 44.6, 45.8, 44.4, 46.4* at 0, 750, 1500 and 2000 ppm, respectively); values were within the range of the historical controls (PND 44.2 - 49.0) and were not dose dependent; the mean weight at attainment was reduced in the 1500 and 2000 ppm group, statistically significant only at 2000 ppm(218.2, 220.4, 207.7, 200.9* g at 0, 750, 1500 and 2000 ppm, respectively). The higher mean age of attainment of balanopreputial separation in the 2000 ppm group was considered to be secondary to lower body weights during pre- and post-weaning periods.
- vaginal patency: age of attainment of the F2 females in the 2000 ppm group was higher than the concurrent control group (mean age at attainment (PN): 33.6, 34.4, 34.4, 35.2* at 0, 750, 1500 and 2000 ppm, respectively); values were within the range of the historical controls (PND 32.5 - 38.8); the authors considered the effect as secondary to lower body weights noted in this group during pre-and post-weaning.
*: statistically significant
- No internal findings that could be attributed to parental exposure to the test article were noted at the necropsies of pups that were found dead or pups weaned at PND 21.
- Pinnal detachment: not affected
- Incisor eruption: not affected
- eye opening: not affected

Effect levels (fetuses)

Dose descriptor:
NOAEC
Remarks:
developmental toxicity
Effect level:
750 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remarks'

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Summary of mean measured test article exposure concentration


Mean nominal concentrations (ppm) by generation

Target concentration (ppm):

0

750

1500

2000

F0 generation

0

749.4

1495.8

2018.1

F1 generation

0

752.8

1495.5

2008.1

F2 generation

0

760.8

1506.3

2009.6

Applicant's summary and conclusion

Conclusions:
Inhalation exposure of rats to 750, 1500 or 2000 ppm n-butyl acetate in a two generation study did not affect reproductive performance. Effects on body weight/body weight gain and food consumption at 1500 and 2000 ppm as well as histopathological changes of the nasal cavity (already at 750 ppm) but no other histopathological effects were observed in parental animals. Adverse effects on pups born to dams exposed to the test article were limited to exposure -related growth retardation (lower mean pup body weights and body weight gains at 1500 and 2000 ppm) and delays in attainment of post-weaning developmental landmarks at 2000 ppm (an effect considered to be secondary to reduced body weight). No teratogenicity was observed. The NOEC for fertility was 2000 ppm, the NOAEC for developmental toxicity 750 ppm and the NOAEC for systemic toxicity 750 ppm.
Executive summary:

A two generation study was performed with rats which were exposed to n-butyl acetate. Four groups of male and female Crl:CD(SD) rats (30/sex/group) were exposed to either clean filtered air or vapour atmospheres of the test article, n-butyl acetate, for 6 hours per day (7 days per week) for at least 70 consecutive days prior to mating (whole body exposure). Target test article concentrations were 750, 1500 and 2000 ppm for the F0, F1and F2 generations. Exposures were initiated when the F0 animals were approximately 7 weeks of age. Exposure of the F0 and F1 males continued throughout mating, and through the day prior to euthanasia. The F0 and F1 females continued to be exposed throughout mating and gestation through gestation day 20. Exposure was suspended from gestation day 21 through lactation day 4 to avoid the confounding effects on nesting and nursing behavior caused by separation of dams from their litters. Therefore, on lactation days 1-4, the F0 and F1 maternal animals received deionized water or the test article via oral gavage at dosage levels of 0, 1125, 2250 and 3000 mg/kg/day (0, 375, 750 and 1000 mg/kg/dose, respectively, administered as 3 equal doses, approximately 2 hours apart), to mimic the internal dose expected from inhalation exposure. Inhalation exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia. Inhalation exposure of the F1 animals was initiated on PND 22 (2 weanlings/sex/litter, when possible), and in order to determine if differences from the control group in mean body weights, body weight gains and food consumption noted in F1 males at 750 ppm during the post-weaning period were reproducible, F1 animals (2 weanlings/sex/litter, when possible) were directly exposed to the test article beginning on PND 22. Following approximately 2-3 weeks of exposure (PND 36-47) of the F1 generation and on PND 36 for the F2 generation, offspring (minimum of 1 animal/sex/litter, to a total of 30/sex/group) were selected for the maturation phase of each generation. F0 males and females were exposed for 113-114 and 121-127 consecutive days, respectively, and F1 males and females were exposed for 134-135 and 140-153 consecutive days, respectively. F2 males and females were exposed for 47-50 consecutive days.

There were no functional effects on reproduction (estrous cycles, mating and fertility indices, number of days between pairing and coitus, spermatogenic endpoints and gestation length) in any test article-exposed group in the F0 or F1 generations. Adverse effects on pups born to dams exposed to the test article were limited to exposure-related growth retardation, as evidenced by lower mean pup body weights, body weight gains at 1500 and 2000 ppm; delays in attainment of post-weaning developmental landmarks were also observed at 1500 and 2000 ppm, which were considered to be secondary to lower body weights noted in this groups during the pre- and post-weaning periods. F1and F2 pup survival was unaffected by the test article at all exposure levels. Therefore, in this study an exposure level of 2000 ppm was considered to be the no-observed-adverse-effect concentration (NOAEC) for fertility and 750 ppm is the NOAEC for developmental toxicity.

General systemic toxicity was evident in the 1500 and 2000 ppm group F0, F1and F2 adult males and females. Decrements in mean body weights, body weight gains and food consumption were observed at 1500 and 2000 ppm. Although body weights, body weight gains and food consumption were reduced in the 750 ppm males of the F1 generation, direct inhalation exposure of F2 animals on a comparable regimen did not reproduce this response. Additionally, the magnitude and temporal pattern of the reductions in the F1males at 750 ppm were not noted in the F0 generation. Therefore, mean body weights, body weight gains and food consumption at 750 ppm were considered to be unaffected by test article exposure. Degeneration of the olfactory epithelium in the nasal cavity was noted at 750 and 2000 ppm in F0 and F1 males and females (endpoint not investigated at 1500 ppm). This finding was considered to be a site-of-contact irritant effect and not indicative of systemic toxicity. Statistically significantly higher mean lung weights (relative to final body weight) in F0 males at 1500 and 2000 ppm, in F1 males at 750, 1500 and 2000 ppm and in F0 females at 2000 ppm and adrenal weights (absolute, relative to brain and/or body weight) in F0 at 1500 and 2000 ppm were noted; all were observed without correlating histologic findings and were considered a stress-related response (adrenal weights) or not adverse (lung weights). Therefore, the NOAEC for systemic toxicity in adult rats was considered to be 750 ppm under the conditions of this study.