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Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: data from a sub-chronic toxicity study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented NTP publication which meets basic scientific principles.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
NTP Technical Report 26 on Toxicity Studies of Ethylene Glycol Ethers 2-Methoxyethanol, 2-Ethoxyethanol, 2-Butoxyethanol Administered in Drinking Water to F344/N Rats and B6C3FX Mice.
Author:
US National Institute of Environmental Scientists
Year:
1993
Bibliographic source:
NIH Publication 93-3349

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD repeat dose toxicity 409
Deviations:
not applicable
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-butoxyethanol
EC Number:
203-905-0
EC Name:
2-butoxyethanol
Cas Number:
111-76-2
Molecular formula:
C6H14O2
IUPAC Name:
2-butoxyethanol
Details on test material:
- Name of test material (as cited in study report): 2-butoxyethanol
- Analytical purity: 99%

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on mating procedure:
not applicable
Duration of treatment / exposure:
13 Weeks
Frequency of treatment:
daily
Details on study schedule:
not applicable
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 750, 1500, 3000, 4500 or 6000 ppm
Basis:
other: These concentrations provided target dose levels of 0, 100, 150, 250, 400 or 650 mg/kg bw per day (US NTP, 1993).
Remarks:
Doses / Concentrations:
0, 69, 129, 281, 367, 452
Basis:
other: actual dose received in mg/kg/day (males)
Remarks:
Doses / Concentrations:
0, 82, 151, 304, 363, 470
Basis:
other: actual dose received in mg/kg/day (females)
No. of animals per sex per dose:
10 male and 10 female rats
Control animals:
yes, concurrent vehicle

Examinations

Oestrous cyclicity (parental animals):
Vaginal cytology was assessed over the 7 days prior to sacrifice. With respect to oestrous cycle evaluations, normally cycling Fischer 344 females have 5-6 day oestrous cycles. While there were 10 females per dose group, 3-4 females per group had estrous cycles longer than 12 days or cycles that were unclear, including the control group. These animals were excluded from the analysis, meaning that only 6-7 females per group were evaluated. Furthermore, given the approximate times in the various stages of estrous (see table below from Zarrow et al., 1964), it appears that the control animals spent an excessive amount of time in estrus. Typically, estrus would comprise approximately 30% of the estrous cycle, rather than the 57.9% reported for the control females. Using Zarrow’s table, if one assumes 2 days for estrus and minimal durations for the other estrous stages, estrus would only comprise 44% of the cycle (2 days out of 4.5 days total estrous cycle). This value is well below the 57.9% reported in the control animals in the study. Vaginal smears were collected only once per day. With only 6-7 rats per group and stages of the cycle that are less than one day in duration, some phases could have gone undetected.

Method: Vaginal vaults were lavaged and the fluid and cells stained with toluidine blue. Relative numbers of leucocytes, nucleated epithelial cells and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (eg. diestrous, proestrus, estrus and metestrus.)
Sperm parameters (parental animals):
Sperm morphology and motility were estimated from epididymal sperm samples taken at necropsy. The left epididymis was isolated and weighed. The tail of the cauda epididymis was then removed and weighed. Test yolk was applied to slides and a small incision made to distal border of epididymis tail. Sperm effluxing was dispersed in buffer on slide and numbers of motile/non-motile spermatazoa counted (5 views per slide.) After this evaluation, epididymis was minced in buffered saline and incubated before sperm density determined (microscopically with a haematometer.) To quantifiy spermatogenesis, testicular spermatid head count was determined by removing tunica albuginea and homogenising left testis in phosphte buffered saline. Homogenisation resistant spermatid nuclei were then counted as for sperm density.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
effects observed, treatment-related

Details on results (P0)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Terminal body weight was decreased by 9 and 14% in males given 4500 and 6000 ppm 2-butoxyethanol. In females, body weights were decreased by 14 and 22% at these concentrations.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no significant differences from the control group in oestrous cycle length for treated females, although females treated at 4500 and 6000 ppm spent more time in dioestrous than the other groups. This correlates with the smaller uterine size, which was attributed to a secondary consequence of reduced body weight gain.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The only spermatozoal measurement that showed significant change relative to the control group was sperm concentration which was slightly decreased in all groups of treated males; however, this effect was not dose-related.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There was no treatment effect on testis weights but there was a reduction in the size of the uterus in females at 4500 and 6000 ppm. There was aslo a decreae in the epididymal weights in the top two dose groups. However, changes in uterine and epididymal weights were considered by the authors to be secondary to the reduction in body weight gain rather than a direct effect of 2-butoxyethanol.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In the rat, haematotoxicity was evident in males at doses of and above 3000 ppm and in all female groups.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
> 452 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: sperm parameters
Dose descriptor:
NOAEL
Effect level:
> 470 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: estrus cyclicity, vaginal cytology.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Data on estrous cycling

Study parameters

0 ppm

3000 ppm

4500 ppm

6000 ppm

n

7

6

7

6

Necropsy body weight (g)1

186 + 4

172 + 2

160 + 2

145 + 2

Estrous cycle length (days)

6.50 + 0.70

6.83 + 0.95

7.57 + 0.53

5.83 + 0.70

Estrous stages (% of cycle)

Diestrus

28.9

45.6

52.8

67.5

Proestrus

8.8

11.4

13.9

7.0

Estrus

57.9

38.6

20.4

17.5

Metestrus

4.4

4.4

13.0

7.9

1 Based on n = 10.

Vaginal cytology during the estrous cycle of the rat1

Time (days)

Stage

Vaginal smear

0.5-1

Proestrus (P)

Many rounded epithelial cells, few leukocytes

1-2

Estrus (E)

Cornified epithelial cells, no leukocytes

0.5-1

Metestrus (M)      

Maximum number of leukocytes, often in clumps surrounding epithelial cells.  For our purposes this will be classified as part of Diestrus.

1.5-2

Diestrus (D)

Few epithelial cells; few to many leukocytes. Sometimes divided into Diestrus I and II

1 Modified from Zarrow et al. (J Endocrinol, 30, 87 -95, 1964).

Applicant's summary and conclusion

Conclusions:
No effects on sperm parameters or estrus cycle/vaginal cytology could be identified that were primarily as a result of treatment with 2-butoxyethanol.
Executive summary:

In a reliable 90 day repeat dose study using oral exposures via drinking water up to 450 -470mg/kg/day in male and female rats, a number of reproductive parameters were examined in the animals that survived to termination. In males, a decrease in epididymal weights was noticed but this was in proportion to overall reduced body weight. Sperm concentrations were also reduced by approximately 5 -10% in all dose groups but there was no dose response relationship evident. There were no effects on estrous length but some evidence that the two highest dose group animals spent more time in the diestrus stage. This correlated with the smaller uterine size, which was attributed to a secondary consequence of reduced body weight gain. In addition, the lack of effect on overall estrous cycle length and absence of histopathologic lesions suggest that the effects on estrous cycle length are unlikely to indicate a specific treatment-related reproductive effect. Thus, the effects on male and female rat reproductive endpoints are not considered to be indications of selective reproductive system toxicity.  The effects seen were minor in nature, not seen in males and only occurred in the presence of other significant systemic toxicity (i.e., decreased body weights, marked haematotoxicity).  In conclusion, the study does not indicate any primary effects on fertility that can be directly attributed to treatment.

Synopsis

NOAEL (sperm effects)>452mg/kg/day

NOAEL (oestrus cyclicity) >470mg/kg/day