Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Jul 1983 - 28 Mar 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Evaluation of a new reproductive toxicology protocol using diethylstilbesterol (DES) as a positive control compound
Author:
Reel, J. R. et al.
Year:
1985
Bibliographic source:
Journal of American College of Toxicology 4: 147-162
Reference Type:
publication
Title:
Assessment of ethylene glycol and monophenyl ether reproductive toxicity using a continunous breeding protocol in Swiss CD-1 mice
Author:
Heindel, J. J. et al.
Year:
1990
Bibliographic source:
Fund Appl Toxicol 15: 683-696
Reference Type:
publication
Title:
Ethylene Glycol Monophenyl Ether
Author:
Lamb, J. C. and Reel, J. R.
Year:
1997
Bibliographic source:
Environmental Health Perspectives 105(1): 225-226
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Reproductive Assessment by Continuous Breeding (RACB); protocol devised by the NTP
Version / remarks:
The RACB protocol does not possess a defined pre-mating exposure. Especially potential effects on male fertility are less likely to become manifest than in the OECD 416 protocol. This deficiency is in part compensated by the cross-breeding (Task 3) which demonstrated the absence of effects on male fertility after a one-week pre-mating exposure.
Deviations:
no
GLP compliance:
yes
Remarks:
Research Triangle Institute
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): Ethylene glycol monophenyl ether (EGPE)
- Physical state:
- Analytical purity: 94-95 % (according to Heindel, J. J. et al., 1990)
- Impurities (identity and concentrations): 6 impurities estimated at a total concentration of 5.5-6.0 % were reported, with none of them exceeding 1.0 %
- Lot/batch No.: C093082 / 01
- Stability under test conditions: Doses were freshly prepared on a weekly basis. Analysis of EGPE in the feed showed a 5.0 % loss in the course of one week.
- Storage condition of test material: stored tightly sealed at 25 °C

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding laboratories Inc., NY
- Age at study initiation: 11 weeks of age at the start of the continuous breeding
- Diet (e.g. ad libitum)
- Water (ad libitum)
- Housing: group housed in solid bottom polypropylene or polycarbonate cages
- Acclimation period: 5 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2
- Humidity (%): monitored by electronic hygrothermographs
- Air changes (per hr): 12-14 times per hour
- Photoperiod (hrs dark / hrs light): 10 /14

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
Each dose of ethylene glycol monophenyl ether (EGPhE) was independently blended into a small amount of ground feed. This mixture was added to a preweighed portion of feed and mixed in a blender for 15 min. Dosed feed was found to lose about 5% of test material over 7 days, therefore, dosed feed was prepared fresh weekly. Concentrations were found to be within 96-105 % of target levels.
Details on mating procedure:
Premating exposure period
Male : 7 days
Female : 7 days
Continuous breeding
Deviation from OECD 416: Differing from the defined pre-mating treatment period laid out in OECD 416, the RACB protocol involves a 98-day continuous breeding period. Unlike the OECD protocol, copulation takes place among animals without appreciable pre-treatment.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis of aliquots of 6 representative samples over the study period revealed EGPE concentrations in the preparations which were within 96-105 % of the expected values.
Duration of treatment / exposure:
7 days prior to and during a 98-day cohabitation period
Frequency of treatment:
Oral feed:
in food, offered ad libitum
Details on study schedule:
Task 1: 14-day dose setting study (range finder)
Task 2: Both sexes were dosed for 7 days prior to and during a 98-day cohabitation period
Task 3: Pairs (P) are mated for 7 days or until a copulatory plug was detected. Treatment was discontinued for all animals during this week then reinstated at the appropriate dose until necropsy (3 weeks after the 7-day cross-over period).
Task 4: the last litter from task 2 is nursed, weaned, reared to sexual maturity while housed by sex two or three per cage and exposed to the same concentration of the test materials as their parents. At 74 ± 10 days of age, males and females from different litters within the same treatment group are cohabited for 7 days or until copulatory plug was seen and then housed individually until delivery. At the end of Task 4, the F1 mice were sacrificed and necropsied.
Doses / concentrationsopen allclose all
Dose / conc.:
0.25 other: % (nominal)
Remarks:
main study (concentrations selected on the basis of range finder)
Dose / conc.:
1.25 other: % (nominal)
Remarks:
main study (concentrations selected on the basis of range finder)
Dose / conc.:
2.5 other: % (nominal)
Remarks:
main study (concentrations selected on the basis of range finder)
Dose / conc.:
1 other: % (nominal)
Remarks:
range finder
Dose / conc.:
2.5 other: % (nominal)
Remarks:
range finder
Dose / conc.:
5 other: % (nominal)
Remarks:
range finder
Dose / conc.:
10 other: % (nominal)
Remarks:
range finder
No. of animals per sex per dose:
Task 1: 8 animals per sex per group
Task 2: 40 breeding pairs for control, 20 breeding pairs per treatment group
Task 3: 3 groups of 20 pairs
Task 4: 19 pairs/group
Control animals:
yes, plain diet
Details on study design:
Animals were 6 weeks old upon receipt and were quarantined for 2-5 weeks. During this period, 2 females and 2 males were killed and their sera was analyzed for 11 viruses. All tests were negative. They were randomly assigned to groups by body weight.

Examinations

Parental animals: Observations and examinations:
Food consumption, parental body weights (time not specified), mortality and clinical signs of toxicity of parents were evaluated.

Oestrous cyclicity (parental animals):
examined
Sperm parameters (parental animals):
testes weight, epididymes weight, enumeration of cauda epididymidis sperm reserve, sperm motility, sperm morphology
Litter observations:
number and sex of pups, live births, live pup weight
Postmortem examinations (parental animals):
Organ weights:
Uterus, ovaries, testes, epididymes (total and cauda), prostate, seminal vesicles, brain, liver, pituitary
Histopathology:
Vagina
uterus
ovaries
oviduct
testis
epididymes
seminal vesicle
prostate
coagulating gland
Postmortem examinations (offspring):
Organ weights:
Uterus, ovaries, testes, epididymes (total and cauda), prostate, seminal vesicles, brain, liver, pituitary
Histopathology:
Vagina
uterus
ovaries
oviduct
testis
epididymes
seminal vesicle
prostate
coagulating gland
Statistics:
The Cochran-Armitage test was used to evaluate any dose-related trends in fertility. A chi square test was used to analyze data from Task 3. Pairwise comparisons between the control and dosed groups were made with the Fisher's exact test. The number of litters and the number of live pups per litter were computed on a fertile pair basis and treatment group means were determined. Dose groups means for these variables, the sex ratio and the proportion of live pups were analyzed using a Kruskal-Wallis test. Ordered differences were tested for by Jonckheere's test.
The Wilcoxon-Mann-Whitney U test was used to make intergroup pairwise comparisons.
An analysis of covariance was performed to correct for the potential effect of the number of pups per litter on the average pup weight. The covariate used was average litter size, including live and dead pups. Least squares estimated of dose group means, adjusted for litter size, were computed and tested for overall equality using an F test and pairwise equality was tested using a t test. Average organ weights adjusted for body weight were tested for equality an analysis of covariance. Absolute organ weights were analyzed by the Kruskal-Wallis and Wilcoxon-Mann-Whitney U tests. Dose-related trends were tested for by Jonckheere's test.

Analyses were performed on data for males and females separately and with both sexes combined. The criterion for significance was p<0.05.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
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:
not specified
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

Task 1: During the 14-days exposure 2 males and 3 females exhibited dehydration in the 7.5 % dose group, one of the females in the 7.5 % dose group died before conclusion of task 1. Three of 7 males and 2 of 4 females in the 10 % group that exhibited dehydration and piloerection during the first week died during the second week. By the Chi-Square and Fischer’s Exact Tests, the percent lethality was significantly elevated in the 10 % dose group. The LD10 and LD50 values were determined to be 8.2 % and 11 %, respectively (Probit Analysis). In addition the % weight gain for the sexes combined was significantly decreased (p = 0.05) in the 5 % group relative to the lower dose and control group. Based on these results dietary levels of, 0, 0.25, 1.25and 2.5 % test substance were selected for Task 2.

Task 2: Continuous exposure of CD-1 mice to these dietary levels had no effect on the number of pairs able to produce at least one litter. No effect on the sex (males/total) of pups born alive was observed. In contrast, exposure to 2.5 % in the feed tended to reduce the number of litters delivered perpair and significantly reduced the average litter size and proportion of pups born alive compared to the control and the 0.25 % and 1.25 % groups. Further there was a significant (p < 0.01) dose-related decrease in live pup weight during continuous exposure of F0 breeding pairs as revealed by Jonckheere’s Test. Live pup weight adjusted for total pups per litter showed a similar dose-related pattern.

Task 3: The proportion of detected matings, fertile pairs pups born alive, sex of pups born alive (males/total) or number of pups/litter did not differ significantly across the three combinations of breeding pairs, absolute live pup weight (sexes combined) adjust for total pups / litter was significantly (p>0.01) decreased for the control males x 2.5 % test substance females versus the control females x control males and 2.5 % test substance males x control females. The results implied that 2-phenoxyethanol was selectively fetotoxic in the F0 females. The control and 2.5 % test substance exposed F0 mice were necropsied three weeks after the 7-day crossover mating period. No significant effects were observed in % motile sperm, sperm concentration, or % abnormal sperm in cauda epididymidis between male control mice and males of the 2.5 % test group. Also no significant effects were observed with respect to brain, pituitary, left testis/epididymes, right testis and prostate weights. However, body weight and seminal vesicle weight were significantly reduced (p > 0.05) and liver weight significantly increased (p > 0.01). When adjusted to body weight the differences in seminal vesicle weight was no longer statistically significant different from control. Liver weight was also significantly increased in female mice fed 2.5 % test substance in the diet compared to control. Body, brain, pituitary, ovary/oviduct and uterine weights did not differ significantly when compared to control.

EGPE reduced reproductive performance at doses that increased liver weights in treated F0 mice.

Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Remarks:
general effects
Effect level:
ca. 3 700 mg/kg bw/day
Sex:
male
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
LOAEL
Remarks:
reproductive effects
Effect level:
> 3 700 mg/kg bw/day
Sex:
male
Basis for effect level:
other: based on the absence of effects on male fertility
Dose descriptor:
LOAEL
Remarks:
general effects
Effect level:
ca. 3 700 mg/kg bw/day
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
LOAEL
Remarks:
reproductive effects
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
general effects
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
male
Basis for effect level:
other: no adverse effects
Dose descriptor:
NOAEL
Remarks:
reproductive effects
Effect level:
ca. 3 700 mg/kg bw/day
Sex:
male
Basis for effect level:
other: no adverse effects
Dose descriptor:
NOAEL
Remarks:
general effects
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
female
Basis for effect level:
other: no adverse effects
Dose descriptor:
NOAEL
Remarks:
reproductive effects
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
female
Basis for effect level:
other: no adverse effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
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:
not specified

Details on results (F1)

Task 4: There was reduced body weight gain to weaning: the middle and high dose groups weighed 25 % and 58 % less than controls at weaning on postnatal day 21; on postnatal day 74, the weight differences were 11 % and 17 %, respectively. Mortality was also increased in the middle and high dose groups from weaning to mating postnatal day 74. This was most pronounced in the high dose group: of the 56 pups weaned in this group, only a total of 6 survived to mating at postnatal day 74. This provided too few animals for statistical analysis. At the mating of the second generation, there was no treatment-related effect on F2 pup number or sex ratio. F2 pup weight adjusted for litter size was reduced in the 1.25 % group by 7 %.
After the delivery of the F2 pups, the control and the 1.25 % group F1 mice were killed and necropsied. The 1.25 % mice weighed 13 % less than controls, their absolute testes weight was 16 % less, and relative seminal vesicles weight was 14 % less than control. The 1.25 % female test group weighed 7 % less than controls; there were no adjusted weight changes in the treated females. There were no treatment-related alterations in epididymal sperm concentration, motility, or morphology.

The test substance caused reduced body weight in neonates in Task 2, 3, and 4, and reduced post-natal survival in F1 animals that were raised to sexual maturity.

Effect levels (F1)

open allclose all
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
male
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 375 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no adverse effects

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
ca. 375 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no adverse effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion