Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
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.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1990
Reference Type:
publication
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Guideline:
other: Reproductive Assessment by Continuous Breeding (RACB); protocol devised by the NTP
Principles of method if other than guideline:
Evaluation of reproductive toxicity of Ethylene Glycol Monophenyl Ether (2-phenoxyethanol) was conducted in Swiss CD-1 mice using the RACB protocol devised by the National Toxicology Program (NTP).
GLP compliance:
yes
Remarks:
Research triangle institute (stated in publication)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Monophenyl Ether (EGPE)
- Analytical purity: 94-95% (with 6 impurities estimated at a total concentration of 5.5-6%, no single impurity present at >1%.

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 (ad libitum)
- Water (ad libitum)
- Housing: group housed in solid bottom polypropylene or polycarbonate cages
- Acclimation period: 5 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 degrees C ±2
- Humidity (%): Not stated in publication.
- Air changes (per hr): Not stated in publication.
- Photoperiod (hrs dark / hrs light): 14/10

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Diet Preperation
EPGE was administered in the feed which was available ad libitum. Each dose was independently blended into a small amount of ground NIH-07 diet (Zeigler Bros, Gardener, PA). The mixture was then added to the pre-weighed portion of the feed and mixed in a patterson kelly 8-quart blender for 15 minutes with the intensifier bar in operation for the first 5 min. Dosed feed was shown to lose about 5% EGPE over 7 days in the cage, therefore dosed feed was prepared fresh weekly. Concentration of EGPE was found to be within 96-105% of the concentration expected within the feed.
Details on mating procedure:
Continuous breeding
- M/F ratio per cage: 1
- Length of cohabitation: 98 days following 7 days of premating dosing with the test article.

The RACB protocol involves a 98-day continuous breeding period, with a 7 day premating dosing period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Aliquots of six representative samples analyzed over the course of the study showed the concentration of EGPE in the preperations to be within 96-105% of the expected values.
Duration of treatment / exposure:
7 day premating and 98 day cohabitation period.
Frequency of treatment:
Administration in oral feed, feed available ad libitum
Details on study schedule:
Task 1: Preliminary dose setting

EGPE was tested at 0.0, 1.0, 2.5, 5.0, 7.5 and 10% in the feed during the 14 day range finding. n = 8/sex/group. Enpoints were clinical signs, body weight and food food consumption.

Task 2: Continous Breeding

The continous breeding phase consisted of a control group (40 breeding pairs), and three dose groups (20 pairs/group). The continous breeding invlolved dosing for 7 days premating and 98 days of cohabitation.

Endpoints observed were: clinical signs, parental body weights, fertility (numbers pairs producing litters/numbers of breeding pairs), litters per pair, lives pups per litter, proportion of pups born alive, sex of live pups, the pup body weights within 18 hours of birth and food and water consumption.

Following the 98 continous breeding period the pairs are seperated and house one animal/cage with continued dosing. Any litter born after the continous breeding are reared by the dam until weaning, after which the chemical is provided at the same concentration as during task 2. The animals are then used for second generation fertility assessment.

Task 3: 1 week mating cross over trial

When a positive effect on fertility is seen during task 2, task 3 is conducted on parental animals after the last litter is weaned to determine the affected sex.

Task 3 consists of three groups of 20 pairs each, control males x control females, control males x high dose females, and control females x high dose males. Pairs are mated for 7 days or until a copulatory plug is detected, whichever is first. Chemical treatment is disconitnued for all animals in this week and then reinstated at the appropriate dose until necropsy.

Endpoints for task 3 are indenitical as task 2. At the end of task 3 F0 males and females are necropsied , the endpoints evaluated are selective organ weight, body weight, epididymal sperm motility, morphology and number, and estrous cyclicity as monitored by vaginal lavage for the preceeding 7 days. Selective organs are examined microscopically after they are fixed in 10% neutral buffered formalin (Bouins fixative for testes), embedded in paraffin and stained with hematoxylin and eosin according to standard procedures.

Task 4: Offspring assessment.

In this phase 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 test chemical in feed 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 is detected, whichever is less, and then housed singly until delivery. The endpoints for this mating trial are the same as those in task 2. At the end of task 4 the F1 mice are necropsied, the endpoints are the same as the necropsy in F0 mice.

Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.25, 1.25, 2.5 %
Basis:
nominal in diet
These concentrations produced calculated consumption estimates of approximately 375, 1875 and 3700 mg/kg/day.
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, concurrent no treatment
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, mortality, clinical signs of toxicity of parents were evaluated, fertility (number of pairs producing a litter/number of breeding pairs).
Oestrous cyclicity (parental animals):
Yes, examined (estrous cycle length)
Sperm parameters (parental animals):
Testes weight, epididymis weight, enumeration of cauda epididymidis sperm reserve, sperm motility, sperm morphology
Litter observations:
Number and sex of pups, live births, live pup weight (within 18 hr of birth)
Postmortem examinations (parental animals):
Organ weights:
Males: testes, epididymis (total and cauda), prostate, seminal vesicles, kidney w/adrenals, liver.

Females: Liver and kidenys w/adrenals.

Histopathology: No details of histopathology are included within the publication.
Postmortem examinations (offspring):
Organ weights:
Males: testes, epididymis (total and cauda), prostate, seminal vesicles, kidney w/adrenals, liver.

Females: Liver and kidenys w/adrenals.

Histopathology: No details of histopathology are included within the publication.
Statistics:
The Cochran-Armitage test was used to evaluate any dose-related trends in fertility (task 2). A chi square test was used to analyze data from Task 3 to establish overall differences in fertility between groups. 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. The proprtion of of live pups was defined as the number of pups born alive , divided by the total number of pups produced by each pair. The sex ratio was expressed as the proportion of male pups born alive out of the total number of live pups born to each fertile pair. Dose groups means for these parameters 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 animals)

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
Gross pathological findings:
no effects observed
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

EGPE was tested at 0.0, 1.0, 2.5, 5.0, 7.5 and 10% in the feed for the dose rangefinding. During this 2 week study, animals in the control, 1.0, 2.0 and 5% groups on average gained 16, 15, 12 and 5% respectively in comparison with their initial weight. In the 7.5 and 10% dose groups, both males and females lost 10% of their initial body weight and three of each sex died. Based on these data a dietary level of EGPE selected for task 2 were 0.0, 0.25, 1.25 and 2.5%.

Task 2

EGPE toxicity was seen in F0 males and female. Toxicity was observed in the form of reduction in body weight (males) and increased liver weights (males and females), there was no significant effect on food consumption during the duration of task 2. Average food consumption was approximately 5.6 g/day/mouse, therefore the daily dose of EGPE for males in the 0.25, 1.25 and 2.5% groups was approxiamtely 375, 1875 and 3700 mg/kg respectively, the daily dose in females varied with the stage of gestation due to fluctuations in body weight.

Continuous exposure of CD-1 mice to dietary levels of 0.25, 1.25 and 2.5% did not affect the number of pairs able to produce at least one litter (fertility index). Exposure to 2.5% EGPE tended to reduce the number of litter/pair and significantly reduced litter size and proportion of pups born alive, the high dose group had 19% fewer live pups/litter compared to controls. These affects were not seen in the 0.25 and 1.25% groups. There was also a significant dose related decrease in adjusted live pup weight during the continous exposure of F0 breeding pairs to EGPE with 4 and 10% reduction in the middle and high dose groups repectively. Examination of litter data for the breeding pairs revealed that in the high dose group only 12 out of 26 (60%) pairs had a fifth litter compared to 36 out of 40 (90%) for control pairs. However the number of pups/litter was not different at 9.8 ± 0.6 for the controls versus 8.8 ± 1.1 for the high dose.

Task 3

The proportion of detected matings, pups born alive, or number of pups/litter did not differ significantly across the three combinations of breeding pairs, absolute live pup weight adjust for total pups / litter was significantly 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 of this crossover study were inconclusive since neither mating nor fertility indices were effected by EGPE pretreatment. Only live pup weight was significantly decreased by 12% in the control x 2.5 % female EGPE mating.

At necropsy 3 weeks after the cross over mating trial there was a signficant decrease in body weight for F0 males (6%) but not for F0 females exposed to 2.5% EGPE in the diet. When adjusted for bodyweight absolute liver weight was significantly elevated in both males and females (14 and 55% respectively). There was no significant differences between control and the 2.5% EGPE males with respect to right testis, prostate and epididymis weights as well as sperm concentration, percentage of motile sperm and percentage of abnormal sperm.

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:
other: based on increased liver weight and reduced body weight.
Dose descriptor:
LOAEL
Remarks:
Reproductive effects
Effect level:
> 3 700 mg/kg bw/day
Sex:
male
Basis for effect level:
other: based on lack of effect on male fertility at all dose levels
Dose descriptor:
LOAEL
Remarks:
General effects
Effect level:
ca. 3 700 mg/kg bw/day
Sex:
female
Basis for effect level:
other: based on increased liver weight
Dose descriptor:
LOAEL
Remarks:
Reproductive effects
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
female
Basis for effect level:
other: based on reduced offspring body weight
Dose descriptor:
NOAEL
Remarks:
General effects
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
male
Dose descriptor:
NOAEL
Remarks:
reproductive effects
Effect level:
ca. 3 700 mg/kg bw/day
Sex:
male
Dose descriptor:
NOAEL
Remarks:
general effects
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
female
Dose descriptor:
NOAEL
Remarks:
reproductive effects
Effect level:
ca. 375 mg/kg bw/day
Sex:
female

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:

Body weights at birth were reduced in the high dose group (~10% reduction compared to controls). 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. Of the 65 pups weaned in the 1.25% group 40 survuved until postnatal day 74. In the high dose group: of the 56 pups weaned only a total of 6 survived to mating at postnatal day 74. This provided too few animals for statistical analysis in the 2.5% group and as such the fertility and reproductive parameters of F1 animals was only assessed in the 1.25% group. At the mating of the second generation there were no statistically significant effects on the proportion of copulatory plug positve mating (mating index), fertile pairs (fertility index), pups born alive or number of pups per litter relative to the F1 controls. As seen with the F0 pairs, live pup weights (F2 pups) were reduced by 7% in the 1.25% group for the F1 pairs. There was also an increase in adjusted liver weight (up 11% in males and 15% in females).

At conclusion of the F1 mating trial the adult mice were necropsied. Body weight was significantly decreased in both males (11%) and females (7%) fed 1.25% groups, while liver weight (adjusted for body weight) was increased compared to the controls. In males at 1.25% absolute testis weight was 16% less and relative seminal vesicle weight was 16% less than controls. There were no effects on right testis, prostate or epdidymal weights or percentage of motile sperm or sperm concentration.

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:
other: based on reduced body weight, increased liver weight, decreased testes weight and increased lethality in pups during lactation.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
ca. 1 875 mg/kg bw/day
Sex:
female
Basis for effect level:
other: based on reduced pup weight and increased lethality in pups during lactation.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 375 mg/kg bw/day
Sex:
male/female

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
ca. 375 mg/kg bw/day
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
2-phenoxyethanol produced reproductive and developmental toxicity at doses that increased liver weight in F0 and F1 mice. 2-phenoxyethanol caused toxicity in growing animals as evidenced by the reduced body weight in neonates in tasks 2, 3 and 4, and the large increase in postnatal lethality as the F1 animals grew to the age of mating.
Executive summary:

In summary 2-phenoxyethanol produced reproductive and developmental toxicity at doses that increased liver weight in F0 and F1 mice. 2-phenoxyethanol caused toxicity in growing animals as evidenced by the reduced body weight in neonates in tasks 2, 3 and 4, and the large increase in postnatal lethality as the F1 animals grew to the age of mating. From this study it was not possible to determine if the increased lethality seen in offspring was caused by quality of milk, lactation problems or EGPE transferred in the milk, however because F1 animals continued to die after weaning it suggests that immature mice are more sensitive to EGPE than the adult.

The data presented for material 2 -phenoxyethanol (EC 204-589-7) is considered appropriate for read-across to EC 202-228-8 and for use in the generation of DNELs and concluding on classification. The justification for the use of this read across is located within IUCLID section 13.