Sodium phenoxyacetate hemihydrate

Toxicity to reproduction

Administrative data

Endpoint:

multi-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983-1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

NTP studies are generally considered to be reliable studies, undergoing a peer review. The NTO Reproductive Assessment by Continuous Breeding (RACB) is based on a multi-generation study comprising a two-generation reproduction toxicity study.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1990

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1997

Reference 3

Reference Type:

publication

Title:

Unnamed

Year:

1984

Report date:

1984

Reference 4

Reference Type:

secondary source

Title:

Unnamed

Year:

2016

Reference 5

Reference Type:

publication

Title:

Unnamed

Year:

1985

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: RACB-protocol of the NTP, as described by Heindel et al. 1990.

Version / remarks:

Reproductive assessment by continuous breeding (RACB)

Deviations:

not specified

Principles of method if other than guideline:

Four tasks were performed. Taken from SCCS 2016:
Task 1:
A 14-day dose finding study (not reported here).
Task 2:
Both sexes are dosed for 7 days prior to and during a 98-day cohabitation period. Animal pairs produce multiple litters during this period. Endpoints include clinical signs, parental body weight, fertility, and food consumption.
Task 3:
When a positive effect on fertility is seen in Task 2, a 1-week crossover trial is conducted in which 20 pairs of parental animals (F0) per treatment group are mated for 7 days or until a copulatory plug is detected. The three treatment groups include: control males x control females, control males x high-dose females, and control females x high-dose males. Treatment is discontinued for all animals during this week then reinstated at the appropriate dose until necropsy (3 weeks after the 7-day cross-over period). At the end of Task 3, F0 males and females are necropsied; endpoints evaluated are selective organ weights, body weight, epididymal sperm motility, morphology and number, and oestrous cyclicity as monitored by vaginal lavage for the preceding 7 days. Selected organs are evaluated for histopathology.
Task 4:
This is conducted whether or not Task 2 shows reproductive toxicity. 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 is seen and then housed individually until delivery. At the end of Task 4, the F1 mice are sacrificed and necropsied.

GLP compliance:

yes

Limit test:

no

Justification for study design:

The RACB protocol was designed and tested by the NTP.

Test material

Test material information

Constituent 1

Reference substance name:

2-phenoxyethanol

EC Number:

204-589-7

EC Name:

2-phenoxyethanol

Cas Number:

122-99-6

Molecular formula:

C8H10O2

Test material form:

solid: particulate/powder

Details on test material:

Batch C093082 / 01

Specific details on test material used for the study:

Abbreviated in the papers to EGPE.
Ethylene glycol monophenyl ether (Cas No. 122-99-6) was obtained from Midwest Research Institute (Kansas City, MO) who also assessed purity and dosing formulation stability. Gas chromatographic analysis indicated that EGPE had a purity of 94-95% with six impurities estimated at a total concentration of 5.5-6%. No single impurity was present at a concentration greater than 1 %.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

COBS Crl:CD-1 (ICR)BR outbred albino swiss mice (CD-1) were purchased from Charles River Breeding Laboratories, Inc., at 6 weeks of age. Mice were 11 weeks of age at the start of the continuous breeding phase of these studies.
On receipt, two males and two females were euthanized and their sera evaluated for antibodies against 11 mouse viruses. All sera were negative for viral antibodies.
After a 2-week quarantine period, some animals were used for the 14-day range finding studies.
All study animals were individually identified by ear tag and assigned to treatment groups using a stratified randomization procedure based on body weights.
Male and female CD-1 mice were group housed by sex during quarantine and during the 1-week premating period they were housed in solid bottom polypropylene or polycarbonate cages with stainless wire lids. The animals were subsequently housed either individually or as breeding pairs.
Ad-Sorb-Dri (Laboratory Products, Inc., Garfield, NJ) bedding was used in all cages.
Deionized filtered water and ground rodent chow (NIH-07) were provided ad libitum. Automatically controlled photoperiods were 14 hr light/ 10 hr dark (lights on from 0700 to 2100 hr), and temperature was maintained at 23 ± 2 °C. Cages were sanitized weekly using detergent and 180°F water. All animal care procedure conformed to the NIH "Guide for the Care and Use of Laboratory Animals", NIH Publication 85-23.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

EGPE was administered in the feed which was available ad libitum. Each dose level was independently blended into a small amount of ground NIH-07 diet (Zeigler Bros., Gardner, PA). This mixture was then added to a preweighed portion of feed and mixed in a Patterson-Kelly 8-quart blender for 15 min 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. Aliquots of six representative samples analyzed over the course of the study showed the concentration of EGPE in the preparations to be within 96-105% of the expected values.

Details on mating procedure:

Task 2. The pre-mating dosing period for males and females was 7 days. Followed by a continuous dosing and continuous mating/breeding for 98 days. Production multiple litters during this period.
Task 3: "A 1-week crossover trial is conducted in which 20 pairs of parental animals (F0) per treatment group are mated for 7 days or until a copulatory plug is detected. The three treatment groups include: control males x control females, control males x high-dose females, and control females x high-dose males. Treatment is discontinued for all animals during this week then reinstated at the appropriate dose until necropsy (3 weeks after the 7-day cross-over period) (taken from SCCS 2016).
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 is seen and then housed individually until delivery. At the end of Task 4, the F1 mice are sacrificed and necropsied. (taken from SCCS 2016).

Duration of treatment / exposure:

7 + 98 + ca. 21 + 74 + ca. 7 + 21 days for animals of tasks 2 and 4.

Frequency of treatment:

Continuous.

Details on study schedule:

See above under mating schedule.
Doses of 0, 0.25, 1.25, 2.5 % were used, corresponding to ca. 400, 2000, and 4000 mg/kg bw/day in males. Daily doses in breeding females calculated from body weight and feed consumption data were approximately double those in males, i.e. the average estimated exposure in females was ca. 950, 4700, and 7500 mg/kg bw/day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 500 mg/kg diet

Remarks:

0.25 %

Doses / concentrations 2

Dose / conc.:

12 500 mg/kg diet

Remarks:

1.25 %

Doses / concentrations 3

Dose / conc.:

25 000 mg/kg diet

Remarks:

2.5 %

No. of animals per sex per dose:

Task 2: 40 males + 40 females in the control; 20 males + 20 females in each test group.
Task 3. 20 males + 20 females in each of the 3 groups.
Task 4: 19 males + 19 females in each group-

Control animals:

yes

yes, concurrent no treatment

Details on study design:

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

Positive control:

No.

Examinations

Parental animals: Observations and examinations:

Clinical signs, parental body weight, fertility, and food consumption.

Oestrous cyclicity (parental animals):

Yes.

Sperm parameters (parental animals):

At necropsy.

Litter observations:

Yes.

Postmortem examinations (parental animals):

Yes.

Postmortem examinations (offspring):

Yes.

Statistics:

Yes.

Reproductive indices:

Yes.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two control mice died, and one mouse and two mice died in the middle and high dose groups, respectively.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not specified

Reproductive function / performance (P0)

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm indices (% motile, epididymal concentration, morphology) were unaffected by EGPE treatment at 2.5%.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Task 2: All pairs of mice in each group had at least 1 litter. There was no reduction in the mean number of litters per pair. The middle dose group had 5.00 litters per pair, while the control had a mean of 4.84; this difference was statistically significant, but biologically insignificant.
The high dose group had 19% fewer live pups per litter than controls; the live pup weight (adjusted for litter size) was reduced by 4 and 10% in the middle and high dose groups, respectively.
Task 3. While there were no alterations in mating or fertility indices or in the number of live pups per litter seen in groups with a treated partner, live pup weight adjusted for litter size was reduced by 12% in the control male x 2.5% EGPE female group.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

12 500 mg/kg diet

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

2

Dose descriptor:

NOAEL

Effect level:

12 500 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Results: P1 (second parental generation)

General toxicity (P1)

Mortality:

mortality observed, treatment-related

Description (incidence):

Task 4. The last F1 litter from all dose levels in Task 2 was reared by the dams to weaning, and then dosed with EGPE at the same concentration provided to their parents. 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 at 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. Because this provided too few animals to analyze, this group was omitted from the rest of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Task 4. The last F1 litter from all dose levels in Task 2 was reared by the dams to weaning, and then dosed with EGPE at the same concentration provided to their parents. 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 at 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. Because this provided too few animals to analyze, this group was omitted from the rest of the study.

Task 4: After the delivery of the F2 pups, the control and 1.25% group F1 mice were killed and necropsied. The 1.25% EGPE mice weighed 13% less than controls, their absolute testis weight was 16% less, and relative seminal vesicles weight was 14% less than controls. The 1.25% EGPE females weighed 7% less than controls; there were no adjusted weight changes in the treated females.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Task 4: After the delivery of the F2 pups, the control and 1.25% group F1 mice were killed and necropsied. The 1.25% EGPE mice weighed 13% less than controls, their absolute testis weight was 16% less, and relative seminal vesicles weight was 14% less than controls. The 1.25% EGPE females 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.

Description (incidence and severity):

Task 4: After the delivery of the F2 pups, the control and 1.25% group F1 mice were killed and necropsied. The 1.25% EGPE mice weighed 13% less than controls, their absolute testis weight was 16% less, and relative seminal vesicles weight was 14% less than controls. The 1.25% EGPE females 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.

Reproductive function / performance (P1)

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Task 4: After the delivery of the F2 pups, the control and 1.25% group F1 mice were killed and necropsied. The 1.25% EGPE mice weighed 13% less than controls, their absolute testis weight was 16% less, and relative seminal vesicles weight was 14% less than controls. The 1.25% EGPE females 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.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

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%, as was the adjusted liver weight (up 11% in males and 15% in females).

Effect levels (P1)

Dose descriptor:

NOAEL

Effect level:

2 500 mg/kg diet

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Results: F1 generation

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 500 mg/kg diet

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Results: F2 generation

General toxicity (F2)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F2 pup weight adjusted for litter size was reduced in the 1.25% group by 7%.

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

2 500 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive toxicity

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

12 500 mg/kg diet

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

For the summary table of the results see the attachment, taken from Lamb et al. 1997.

EGPE was not particularly toxic to either male or female CD-1 mice since no males and only three females died during Task 2. This low general toxicity was manifested by only a minimal effect of EGPE on male body weight (2% decrease) with no average change in female weight.

There was no significant effect of EGPE on feed consumption during the 14 weeks of Task 2. The average feed consumption was approximately 5.6 g/day/mouse.

Therefore, the daily dose of EGPE for the males in the 0.25, 1.25, and 2.5% groups was approximately 0.4, 2.0, and 4.0 g/kg body wt, respectively: the daily dose of the females varied with the stage of gestation due to fluctuations in body weight.

Continuous exposure of CD-1 mice to dietary EGPE at 0.25, 1.25, and 2.5% had no effect on the number of pairs able to produce at least one litter (fertility index). Exposure to 2.5% EGPE, but not to 1.25 or 0.25%, tended to reduce the number of litters delivered per pair and significantly reduced the litter size and proportion of pups born alive.

Further, there was a significant dose-related decrease in adjusted live pup weight during continuous exposure of F0 breeding pairs to EGPE. A more detailed examination of the litter data for the breeding pairs revealed that in the high-dose group only 12 out of 20 ( 60%) pairs had a fifth litter compared to 36 out of 40 (90%) for control pairs. However, the number of pups/litter for the fifth litter was not different at 9.8 ± 0.6 for the controls versus 8. 8 ± 1.1 for the high-dose group.

The results of the crossover mating were inconclusive since neither mating nor fertility indices were altered by EGPE pre-treatment. Only live pup weight was significantly decreased by 12% in the control X 2.5% female EGPE mating.

At necropsy 3 weeks after the crossover mating trial there was a significant decrease in body weight for F0 males but not for F0 females exposed to 2.5% EGPE in the diet. When adjusted for body weight, liver weight was significantly elevated in both males and females (21 and 60%, respectively). There were no significant differences between the control and the 2.5% EGPE males with respect to right testis, prostate, and epididymal weights as well as sperm concentration, percentage of motile sperm, and percentage of abnormal sperm.

In order to assess the reproductive effects of EGPE on the F1 generation, the final Task 2 litters from the 0, 0.25, 1.25, and 2.5% EGPE groups were weaned at 21 days of age and 8 to 10 litters per group were randomly selected for rearing. At 74 ± 10 days of age one to three female and male pups from each surviving litter were randomly selected for breeding within their treatment group; sibling matings were avoided.

Body weight at birth (Day 0), weaning (Day 21 ), and mating (Day 74 ± 10) showed dose-related decreases, indicating EGPE toxicity during the lactation and post-weaning periods. Moreover, pup lethality was pronounced in the 1.25 and 2.5% EGPE groups during the lactation (Days 0 to 21) and post-weaning periods (Days 21 to 74 ± 10). By Day 21 only eight litters in these two groups had a sufficient number of male and female pups (at least one of each sex per litter) for the subsequent mating trial. From birth to 74 ± 10 days of age, 12 out of 87 (14%) of the selected offspring died in the control group, 20 out of 113 ( 18%) in the 0.25% EGPE group, 33 out of 84 (39%) in the 1.25% EGPE group, and 66 out of 76 (87%) in the 2.5% EGPE group. Because of the high lethality rates (25 out of 32 males and 21 out of 24 females) in the F1 weanlings exposed to 2.5% EGPE in the diet from Days 21 through 74 ± 10, only three pairs were available for breeding at Day 74 ± 10. Therefore, F1 matings were conducted with the control and 1.25% EGPE groups. Continuous exposure of the F1 mice to 1.25% EGPE indirectly in utero and during lactation and directly from weaning to 74 ± 10 days of age had no statistically significant effects on the proportion of copulatory plug positive matings (mating index), fertile pairs (fertility index), pups born alive, or number of pups per litter relative to the control F 1 mice. As observed earlier for the F0 pairs, live pup weight (F2 pups) was diminished for the F1 pairs.

At the 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% EGPE, while liver weight (adjusted for body weight) was increased compared to that of controls.

There was no effect of EGPE on right testis, prostate, or epididymal weights or on percentage of motile sperm, percentage of abnormal sperm, or sperm concentration. Seminal vesicle weight was decreased significantly relative to that of controls.

Applicant's summary and conclusion

Conclusions:

In summary, ethylene glycol monophenyl ether (PhE) produced significant reproductive and developmental toxicity at doses that increased liver weight in treated F0 and F1 mice. Ethylene glycol monophenyl ether caused significant 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.
The results indicate that PhE adversely affects fertility as well as other reproductive parameters in mice in the presence of general toxicity.

Executive summary:

From Heindl et al. 1990: "A continuous breeding reproduction study design was utilized to examine the reproductive toxicity of ethylene glycol monophenyl ether (EGPE; 2-phenoxyethanol; PhE). Swiss CD-1 mice were used. EGPE was administered via the feed (0, 0.25, 1.25, and 2.5%, i.e., 0, 0.4, 2.0, and 4 g/kg body wt/day). Both male and female mice were dosed for 7 days prior to and during a 98-day cohabitation period.

With EGPE, there was no change in the ability to produce five litters during the continuous breeding period. There was, however, a significant but small ( 10-15%) decrease in the number of pups/litter and in pup weight in the high-dose group. A crossover mating trial suggested a female component of the reproductive toxicity of EGPE. While fertility was only minimally compromised, severe neonatal toxicity was observed. By Day 21 there were only 8 out of 40 litters in the mid- and high-dose groups which had at least one male and female/litter. Second generation reproductive performance of the mid-dose group (1.25%) was unaffected except for a small decrease in live pup weight.

In summary the reproductive toxicity of EGPE was only evident in the female and occurred at doses which elicited general toxicity. EGPE was particularly toxic to immature mice of both sexes."

Taken from SCCS (2016): "In this study, fertility was only minimally affected at the highest dose, but evidence of significant toxicity to the offspring was observed when 2-phenoxyethanol was administered at 1.25% and 2.5% in diet. The cross-over mating trial suggested a female component to the reproductive toxicity observed in high-dose females. The authors concluded that for male mice (F0) the NOAEL for reproductive toxicity was 2.5% in diet, corresponding to 4000 mg/kg bw/day, and this occurred in the presence of evidence of parental toxicity (decreased body weight and increased liver weight).

For both males and females, the NOAEL for parental toxicity and reproductive toxicity was concluded to be the low dose, i.e., 0.25% in diet. For males, a NOAEL of 400 mg/kg bw/day was calculated. The estimated corresponding daily intake of 2-phenoxyethanol in females calculated from average body weight and average feed consumption reported during week 18 was approximately 950 mg/kg bw/day."