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Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Well documented publication which meets basic scientific principles”. Full study report also available

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Association of sperm, vaginal cytology and reproductive organ weight data with results of continuous breeding reproduction studies in Swiss CD1 Mice.
Author:
Morrissey RE , Lamb JC, Schwetz BA, Teague JL and Morris RW
Year:
1988
Bibliographic source:
Fundamental and Applied Toxicology, 11, p359-371.
Reference Type:
publication
Title:
Results and evaluations of 48 continuous breeding reproduction studies conducted in mice.
Author:
Morrissey RE , Lamb JC, Morris RW, Chapin RE, Gulati DK and Heindel JJ
Year:
1989
Bibliographic source:
Fundamental and Applied Toxicology, 13, p747-777.
Reference Type:
publication
Title:
Assessment of Ethylene Glycol Monobutyl and monophenol Ether reproductive toxicity using a continuous breeding protocol in Swiss CD-1 mice.
Author:
Heindel JJ , Gulati DK, Russel VS, Reel JR, Lawton AD and Lamb JC
Year:
1990
Bibliographic source:
Fundamental and Applied Toxicology, 15, p683-696.
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report date:
1985

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: National Toxicology Programme Continuous Breeding Protocol
Deviations:
yes
Remarks:
, F1 tests were only carried out with the control and low dose groups
GLP compliance:
yes
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): ethylene glycol butyl ether
- Analytical purity: >99% (confirmed by GC)
- Water 0.14% (by Karl Fischer
- Supplier: Midwest research inst, Kansas City.

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston NY
- Age at study initiation: (P) x 11wks; (F1): 74+/-10 days
- Housing: by sex in solid bottom polypropylene or polycarbonate cages with stainless steel wire lids, then subsequently in breeding pairs. Ad-Sorb-Dri bedding used.
- Diet (ad libitum): NIH-07
- Water (ad libitum): tap
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Photoperiod (hrs dark / hrs light): 10/14

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Each dosing solution formulated directly. No loss of compound after 3 days at room temperature and only 0.9% after 21 days lost. Consequently, fresh solutions prepared every 2 weeks.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 7-day pre-mating period and a 98-day cohabitation period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis on days 1, 6, 10 and 14. All doses found to be within 97-104% of expected values.
Duration of treatment / exposure:
1 week initially, 14 weeks during cohabitation, then 3 further weeks.
Frequency of treatment:
continuous
Details on study schedule:
At the completion of the continuous breeding phase, the F0 breeding pairs were separated and housed individually and exposure to 2-butoxyethanol continued. When the last litter was weaned from the continous breeding phase F0 males and females from the 1 % dose group were mated with male and female control animals in a one-week crossover mating study to determine any sex-related reproductive effects of 2-butoxyethanol.

Doses / concentrationsopen allclose all
Dose / conc.:
720 mg/kg bw/day (actual dose received)
Remarks:
0.5% nominal in drinking water
Dose / conc.:
1 340 mg/kg bw/day (actual dose received)
Remarks:
1% nominal in drinking water
Dose / conc.:
2 050 mg/kg bw/day (actual dose received)
Remarks:
2% nominal in drinking water
No. of animals per sex per dose:
20. 40 in control
Control animals:
yes, concurrent no treatment
Details on study design:
-The study itself consisted of three phases:
Phase 1 = continous breeding phase
Phase 2 = F0 males and females mated with control animals
Phase 3 = F1 males and females mated

Exposure to 2-butoxyethanol was discontinued during the one-week mating period and then reintroduced at 1 % dose level (estimated daily intake 1830 mg/kg bw). Control males and females were also mated for comparative purposes. The proportion of successful copulations from the breeding pairs was similar in all groups. However, the number of fertile females was significantly reduced in the group where treated females were mated with control males.

A final phase was conducted to assess the fertility and reproductive effects of 2-butoxyethanol in second generation (F1) pups. The pups selected were those born after the CBP and when the maternal animals were individually housed. As there were insufficient pups in the 1 and 2 % dose groups, only the pups from the 0.5 % dose group were used. The F1 generation pups were nursed, weaned and reared to sexual maturity. After weaning, the mice received 0.5 % 2-butoxyethanol in their drinking water (estimated daily intake 950 mg/kg bw). At 74 ± 10 days of age, the F1 animals from different litters were mated. The animals were necropsied after delivery.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS AND CLINICAL OBSERVATIONS: Yes
- Time schedule: twice per day
- Cage side observations: loss of hair, diarrhea, wounds, activity, occular effects, paralysis, nasal discharge, blood in cage, posture, death.

BODY WEIGHT: Yes
- Time schedule for examinations: 7 days before start, start then days 7, 28, 56, 84, 112

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily
Oestrous cyclicity (parental animals):
F0 Females Mated with Control Males
-When evaluated over the 7 day period prior to necropsy, proportionally more females (7/13) in the 1 % treated group than controls (9/38) had oestrus cycles longer than 7 days.
Sperm parameters (parental animals):
Parameters examined in P and F1 male parental generationsParameters examined in P and F1 male parental generations according to the NTP sperm morphology vaginal cytology evaluation protocol: covers eg. sperm count, sperm motility, sperm morphology.
Litter observations:
Parameters examined in P and F1 generationsSTANDARDISATION OF LITTERS
- Performed on day 4 postpartum: not specified

PARAMETERS EXAMINED
The following parameters were examined in offspring: pup body weight, sex ratio, litters per pair, live and dead pups

GROSS EXAMINATION OF DEAD PUPS: no data

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
HISTOPATHOLOGY / ORGAN WEIGHTS
Liver, brain, pituitary, testicle, prostate, seminal vesicle, epididydimus, complete femal reproductive tract
Postmortem examinations (offspring):
The protocol only requires this if there is evidence of reprotoxicity. In this case there was no justification for postmortems on the offspring so this was not carried out other than organ weights.
Statistics:
Cochran-Armitage test for dose related trends in fertility. Chi-square test for differences in fertility among groups. Pairwise comparisons between dose groups and control using Fisher's exact test. Reproductive indices between groups compared using Kruskal-Wallis test and ordered differences using Jonckheere's test. Pairwise comparisons of treatment groups perfomed using Wilcoxon-Mann-Whitney U test. Pup number per litter corrected for when calculating average pup weight.
Reproductive indices:
The numbers of fertile pairs from the surviving pairs of the continuous breeding phase were 38/39, 19/19, 13/14 and 5/7 at 0, 0.5, 1.0 and 2.0 % dose levels, respectively.
Offspring viability indices:
no further information

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Description (incidence):
During the 98 day cohabitation period, deaths occurred in the female mice: 13/20 in the 2 % group, 6/20 in the 1 % dose group, 1/20 in the 0.5 % dose group and 1/40 in the control group. In the male mice, no deaths occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The average body weights in the female 2 % dose group were consistently lower than the controls (worst at week 10 where it was -36%). The females in this group also showed weight loss between weeks 1-3. Male groups experienced weight loss (1-2 % of initial body weight) in the two highest doses.
At necropsy, male and female mice from the 1 % dose group had significantly lower body weights (females -10%, males -7.4%)
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Reduced fluid consumption was observed at all dose levels in both sexes (22 %, 18 % and 36 % reduction relative to controls at 0.5 %, 1.0 % and 2.0 %, respectively after 14 weeks of dosing).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In the only histopathological examination carried out on the treated females, no treatment related kidneys lesions were observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
In the 1% group no significant differences were observed between the control and treated animals for the average oestrous cycle length and frequency.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
-In the 1% group no significant differences were observed between the control and treated animals for the weights of male reproductive organs, sperm motility or morphology.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Significant reduction in reproductive performance occurred at 1 and 2 % dose levels as indicated by dose-related decrease in number of litters per fertile pair and litter sizes (mid dose group -21%, high dose group -53%). The number of live pups per litter was also reduced in these two dose groups (mid dose: -52%, high dose -70%) as was the proportion of pups born alive (mid dose -37%, high dose -44%). The fertility index was significantly reduced at 2% although care is required in the interpretation of this result due to the reduced number of animals caused by the severe toxicity and mortality seen in this dose group.

Details on results (P0)

Based on the fertility index and pup weights, the crossover trial (1% dose animals versus controls) suggested that the reduced fertility at this dose was more due to toxicity to females than males.

Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Effect level:
720 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
water consumption and compound intake
Dose descriptor:
NOAEL
Effect level:
720 mg/kg bw/day
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
organ weights and organ / body weight ratios
reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A significant increase in relative kidney weight in the females, and a significant increase in relative liver weight in both the males and females were observed in the P1 generation.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No treatment-related changes in the oestrous cycle length and frequency were noted in the P1 generation.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No treatment-related changes in the weights of reproductive organs, sperm motility or morphology were noted in the P1 generation.
Reproductive performance:
no effects observed
Description (incidence and severity):
No significant fertility and reproductive effects were observed in the P1 animals as indicated by the proportions of successful copulation and fertile females, litter size, pup viability and live pup weights.

Details on results (P1)

On the P1 generation (low dose and control only tested) , there were no effects on mating and fertility indices or any other reproductive parameter. There were no effects on sperm parameters in males or estrous cyclicity in females but relative liver and kidney weights were increased in the dose group receiving 2-butoxyethanol (0.5%)

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
720 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity (P1)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Description (incidence and severity):
-Significant reduction in reproductive performance occurred at 1 and 2 % dose levels as indicated by dose-related decrease in pup viability.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Effects were seen in all dose groups. Adjusted pup weights were reduced as follows low dose group -3%, mid dose group -10%, high dose group -15%. Live pup weights unadjusted were reduced similarly.
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
720 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: pup weight

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effect was seen in the pups born to the P1 generation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increase in kidney weight at 0.5% dose (+22%)

Details on results (F2)

There were no significant differences in F2 litter size, pups numbers or weights.

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
720 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects seen at the only tested dose

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

The continuous breeding first generation study showed that treatment significantly affected fertility at the mid and high dose levels and marginally affected pup weight at the lowest level. The cross-breeding results suggest that the fertility effects were only due to effects on the female mice. In the cross-over study there were no effecting on mating index (number with copulatory plugs/number co-habited), however for females that co-habited with control males the fertility index (number fertile/number with copulatory plugs) ws significantly reduced compared to the controls and other cross-over group. Similarly, the number of live pups/litter of the 1% treated females/control males fell. These effects may have been an indirect consequence of the severe general systemic toxicity rather than a direct effect of 2-butoxyethanol on the reproductive organs.

Applicant's summary and conclusion

Conclusions:
In this study, effects were seen on fertility only at doses which were severely toxic to the animals (1340 and 2050 mg/kg bw/d). A NOAEL of 720 mg/kg bw/day can be identified for fertility effects by oral route in mice. Marginal pup weight reductions at this dose were not repeated in the second generation and therefore not regarded as a significant finding.
Executive summary:

In a continuous breeding study carried out to an NTP protocol, mice were exposed for a period of 14 weeks to the oral drinking water routes at doses from 720 to 2050mg/kg. Significant adverse reproductive effects were observed in the females at very high dose levels (1340 mg/kg and above) which also caused severe general toxicity, including death. Under the conditions of the study, the NOAEL for reproductive toxicity of 2-butoxyethanol (fertility) can be set as 720 mg/kg/day. For developmental toxicity, no NOAEL can be derived. A conservative LOAEL of 720 mg/kg/day can be taken as only a very slight decrease in pup weight was observed at this dose.

At the lowest dose studied, the only adverse finding was a marginal statistically significant reduction in pup weight. However, since this reduction was only 3% compared to controls and was not repeated in the F1 generation, it was not considered a significant adverse finding.

No NOAEL or LOAEL can be determined for systemic parental toxicity because this kind of study is not designed to assess systemic toxicity although it is of note that there were effects (reduced fluid consumption) even at the lowest dosage of 720 mg/kg/day.