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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This was a study carried out to assess whether a recognised protocol for mice could be used with rats. A full and comprehensively documented study report is available. Whilst the study is not documented as compliant with GLP, it has been subject to rigorous QA comparative to the GLP process. The study does omit some aspects of the study that would be included if the toxicity of the substance was unknown, but all the end points likely to be important for this substance were examined.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1990
Reference Type:
publication
Title:
Reproductive assessment by continuous breeding: evolving study design and summaries of ninety studies,
Author:
Chapin RE, Sloane RA
Year:
1997
Bibliographic source:
Env Hlth Perp, 105 Suppl 1, 199-205

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: NTP continuous breeding protocol
Deviations:
yes
Remarks:
rats used instead of mice
Principles of method if other than guideline:
Reproductive assessment by continuous breeding design (RACB). Purpose of study was to develop RACB mouse protocol for use with rats using a known toxicant. Full method described in Chapin (1997) – see reference list. In a modification of the standard protocol, rats were cohabited for ~6 weeks, separated for delivery, nursing and weaning of the second litter for the F2 study, then cohabited for another ~9 weeks to produce a further 3 litters for other activities e.g. cross over mating. Cross over mating was used to determine the affected sex.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: liquid
- Analytical purity: 99.9% pure
- Lot no: CMB, MRI batch #01
- Composition: Purity determined by GC >99%
- Other: Stability confirmed when stored at up to 60C for 14 days and no measurable loss from 5% solutions.
- Source: General Drug and Chemical Co, via Midwest Res. Inst, Kansas City, MO.

Test animals

Species:
rat
Strain:
other: Sprague Dawley VAF Crl:CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Housing: Two per cage during quarantine and 1 week premating, then breeding pair or individually. Solid bottomed polycarbonate cages. Sani-chip bedding. Cages disinfected weekly.
- Diet: Pelleted rodent lab chow (NIH-07) ad libitum
- Water: deionised, available ad libitum
- One male, one female checked negative for antibodies to 7 rodent viruses, m. pulmonis plus CAR Baccillus.

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/-3
- Photoperiod: 14 hours dark/10 hours light

Administration / exposure

Route of administration:
oral: drinking water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Mixed with deionised water on a weight/volume basis.
Details on mating procedure:
Males and females cohabited for approximately 6 weeks (a deviation from the standard protocol) to allow delivery, nursing and weaning of the 2nd litter, then re-co-habited for approximately nine more weeks.
M/F ratio: 1 to 1. F1 pairs cohabited for 1 week.
Length of cohabitation: 6 week period then 9 week period to produce 3 more litters.
Otherwise standard RACB protocol.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Bulk solution every 4-6 weeks. Bulk solutions confirmed stable for this period. Dosing solutions prepared at max interval of 3 weeks and stored at room temperature and normal lighting. Doses were in the range 93-103% of target dose.
Duration of treatment / exposure:
Continuous from 1 week before mating through F1 generation.
Doses / concentrations
Remarks:
Doses / Concentrations:
0.01%, 0.03% and 0.1% (F0 average equivalents 11mg/kg, 30mg/kg, 100mg/kg, F1 average equivalents 12mg/kg, 34mg/kg – low and mid dose groups only.)
Basis:

No. of animals per sex per dose:
20 pairs per dose group. 40 in control.
Control animals:
yes
Details on study design:
Dosing for 1 week prior to pairing. F1 pups weaned on postnatal day 21 and weighed weeks 21-23. 2nd generation from 2nd litter of F0 animals.
Dose selection rationale: data from previous studies. Top dose designed.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: weeks 1-6, 10, 15, 18 and after every litter. F1 animals at weaning and weeks 31-33.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Calculated based on data from weeks when body weight and consumption measured on a per cage (per breeding pair) weekly average.
Oestrous cyclicity (parental animals):
Yes. Relative frequency of oestrus, proestrus, metestrus, diestrus, cycle length.
Sperm parameters (parental animals):
Parameters examined included motility, density, and percent abnormal. Sperm analysis is contained in a separate appended report to the main reference.
Litter observations:
-Drinking water consumption monitored week 21. Body weights. F2 pups monitored for 21 days (on days 0, 4, 7, 14, 21).
- standardization of litters: No
- parameters examined: litters per pair, live pups/litter, proportion of pups born alive, sex ration, live pup weight, (adjusted and unadjusted.)
- Gross examination of dead pups:
Postmortem examinations (parental animals)
- Sacrifice: Male animals: At the end of the crossover examination. Maternal animals: not examined.
- Gross necropsy: Yes
- histopathology/organ weights: liver, kidneys, right testis and epididymis and cauda epididymis, seminal vesicles and prostate, ovary. Histopathology only on males in the control and two lower dose groups as previous data indicated no effects likely on females. 10 randomly selected animals per dose group per generation examined. Livers only examined if gross effects seen.
Postmortem examinations (offspring):
HISTOPATHOLOGY / ORGAN WEIGTHS
F1 animals
Statistics:
Cochran Armitage for dose related trends in fertility. Chi square test for cross over mating. Fisher’s exact test for pairwise comparisons with controls. Kruskal-Wallis (KW) and Jonckheere’s test for fertility parameters. Pairwise comparisons between treatment group means using Wilcox-Mann-Whitney U (WMWU) test. Average pup weight corrected for number of pups per litter using analysis of covariance; organ weights adjusted for total body weight similarly. Unadjusted body/organ weights analysed using KW and WMWU test and dose related trends tested using Jonckheere’s test. Latter also used to check body weight and water consumption data with significance among dose groups assessed using Shirley’s test when a trend in evident or Dunn’s test when one is not.
Reproductive indices:
Yes. Number of litters and live pups per litter, proportion of live pups, sex ratio. Mating index.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights were significantly reduced from week 3 in females and week 6 in males in the high dose group only. Water consumption declined from week2 onwards in both sexes in the high dose group. It was significantly reduced in the mid dose group in week 6 only (4 other measurements not significantly different.)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No significant effects.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Motility and density were decreased in the top dose group. Motility increased marginally but by a statistically significant margin in the mid dose group. All parameters were normal in the low dose group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
100% fertility in controls and low dose group, 89% in mid dose group and only 5% (significantly lower – single breeding pair) in the top dose group. Days to litter were similar in the control and low dose group but increased in the mid dose group. (Too few in high dose group.). The number of litters per pair was not affected in the low and mid dose group. But the number of live pups per pair was significantly reduced in the mid dose group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
All significantly reduced in the high dose group. In the mid dose male group adjusted liver seminal vesicle and epididymides weights significantly reduced. In the low dose group, the adjusted prostate weight increased significantly. No changes were seen in the mid dose female group.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Testicular and epididymal lesions seen, but also present in controls. Effects were not attributed to treatment.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
0.01 other: % (11mg/kg)
Sex:
male
Basis for effect level:
other: Adverse effects on sperm and male reproductive organs.

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Few pups born to high dose animals. Live pups/litter and proportion of live pups/litter decreased significantly in the mid dose group. The proportion of still births, the average postnatal survival were significantly decreased in the mid dose group.

BODY WEIGHT (OFFSPRING)
Mean body weights of mid dose pups were significantly reduced for both sexes at all time points except one. Adjusted live pup weight increased significantly in the low dose group albeit by only around 5%. F1 mature animals showed reduced body weight (-14%) in the mid dose group.

SEXUAL MATURATION (OFFSPRING)
No adverse changes on reproductive performance.

ORGAN WEIGHTS (OFFSPRING)
There was no effect on adjusted organ weight of females. In males, liver, kidney, seminal vesicle, cauda epididymis, right epididymis and prostate all showed lower weights in the mid dose group after adjustment. The low dose group animals were similar to controls

HISTOPATHOLOGY (OFFSPRING)
Only the testes and epididymis were examined. Minimal to mild testicular lesions were seen in 2/10, 7/10, 5/10 animals in the controls/mid/high dose animals respectively. These results suggested a slight effect from treatment but the no clear dose response was evident. Single animals in the mid and high dose animals showed epididymis lesions, which were not attributed to treatment.

OTHER FINDINGS (OFFSPRING)
Drinking water consumption decreased in the mid dose group from week 21. The estimated daily dose was 9 and 27mg/kg for the low and mid dose group male pups and 15/41mg/kg respectively for females. There was no effect the litter indices of either F1 dose group animals. There were no differences in epididymal sperm morphology or motility among the groups from the F2 generation but there was significantly decreased sperm density in both dose groups (-17% in low dose group, -23% in mid dose group).

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
0.01 other: % (11mg/kg)
Sex:
male/female
Basis for effect level:
other: Reduced number of live pups per litter.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

Methoxyethanol when administered to SD rats over 2 generations and dosed via drinking water was toxic to reproduction.  The top 0.1% dose almost completely inhibited reproduction in the F0 generation and the mid dose 0.03% significantly reduced the number of live pups borne; other adverse changes were seen at this dose on male testes and sperm. The lowest dose 0.01% (equivalent daily dose ~10mg/kg) showed minor changes to sperm density and testicular lesions, but these could not be clearly attributed to treatment with a clear dose response relationship and therefore not considered significant adverse effects.