Sodium phenoxyacetate hemihydrate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A read-across to a multigeneration study with 2-phenoxyethanol (PhE) in mice is performed. PhE is the metabolic precursor of phenoxyacetate, which is the common anion to the target substance sodium phenoxyacetate and also to phenoxyacetic acid. A read-across analogue approach is described and attached to the target record.

In summary, the reproductive toxicity of PhE was only evident in the female and occurred at doses which elicited general toxicity. 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, for females a higher NOAEL, corresponding to the daily intake was estimated.

Link to relevant study records

Reference

Reference 1

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.

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.

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 %.

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.

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

Dose / conc.:

2 500 mg/kg diet

Remarks:

0.25 %

Dose / conc.:

12 500 mg/kg diet

Remarks:

1.25 %

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.

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.

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: 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.

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

Dose descriptor:

NOAEL

Effect level:

12 500 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

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: 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).

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

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

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%.

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

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

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.

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."

Reference 2

Endpoint:

multi-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The sodium phenoxyacetate metabolic pathway analogue approach is presented.
The description of this analogue approach - in a more extensive and structured form - is attached.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
1.1. Analogue Hypothesis
The target substance (to be registered) is sodium phenoxyacetate (NaPhA). The source substance is phenoxyethanol. Phenoxyethanol is the metabolic precursor of phenoxyacetate.
The hypothesis is that systemic toxicity caused by oral dosing of PhE to mammals comprise at least the toxicity that would be obtained after oral dosing of NaPhA. The dose descriptor NOAEL for PhE can be transcribed as a worst case to NaPhA.
For more details see the attached justification.

1.2. Applicability domain (AD) of the analogue approach
The approach is suitable for read-across of systemic effects observed in toxicity studies with mammals. Primarily oral toxicity studies are concerned, 1) because the practically complete absorption of the source and the target substance is known, and 2) because haematotoxicity, caused by the not yet metabolised source substance PhE, was observed especially after dermal exposure to rabbits.
The applicability of the approach to toxicity endpoints using the dermal or the inhalation route needs some caution.

The read-across approach is not suitable for ecotoxicity endpoints, because PhE is readily biodegradable to CO2 in aqueous media and no stable metabolite PhA- is formed in ecosystems as in the case of mammalian systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Sodium phenoxyacetate
Synonyms: Acetic acid, 2-phenoxy-, sodium salt (1:1); etc.
Abbreviated to: NaPhA (or NaPhAA)
CAS-No.: 3598-16-1
Molecular Formula: C8H7NaO3
Molecular mass: 174.13
Smiles Code: c1(OCC(=O)[O-])ccccc1.[Na+]
Physical state: Solid.

Phenoxyacetic acid
Abbreviated to: PhAA
CAS: 122-59-8
Molecular formula: C8H8O3
Molecular mass: 152.149
SMILES: O=C(O)COc1ccccc1
Physical state: Solid.

NaPhA and PhAA are dissociated in water at pH > ca. 4. Therefore the common anion phenoxyacetate (PhA-) and the cations Na+ and H+ also occur.

Phenoxyethanol
Synonyms: 2-phenoxyethanol; ethyl glycol monophenol ether; ethylene glycol monophenyl ether; phenoxytol; 1-hydroxy-2-phenoxyethane; (2-hydroxyethoxy) benzene
Abbreviated to: PhE; (PE)
CAS-No.: 122-99-6
Molecular Formula: C8-H10-O2
Molecular mass: 138.165
Smiles Code: c1(ccccc1)OCCO
Physical form: Oily, slightly viscous liquid at room temperature

2.1. Purity / Impurities
- NaPhA: The registered substance has a purity >96 %. Impurities are sodium chloride and water. Purity is therefore not a relevant factor for the analogue approach, to provide data for systemic toxicity endpoints only.
- PhAA: Purity is not relevant, as PhA- is the major metabolite of PhE and no studies will be proposed with PhAA. Purity is therefore not a relevant factor for the analogue approach.
- PhE: The analytical purity of the test substances used for the key studies on repeated dose toxicity and developmental toxicity were 99.9 % or above. The purity is considered high enough to not impede the analogue approach.


3. ANALOGUE APPROACH JUSTIFICATION
Supporting physical-chemical properties, toxicokinetics and metabolism of the analogues
For more details see the attached justification.
Conclusion:
Phenoxyacetic acid and sodium phenoxyacetate are easily and practically completely absorbed after oral administration. The common anion phenoxyacetate is excreted practically completely and unchanged in urine.
Phenoxyethanol PhE is rapidly and rather completely absorbed after oral dosing. PhE is rapidly metabolised predominantly to PhA- and rapidly excreted mainly as unchanged PhA-. More than 90 % of an oral dose of 2-phenoxyethanol was excreted in the urine of rats as phenoxyacetic acid within 24 h.
PhE is well absorbed through the skin, but not completely. Remarkable is that PhE is metabolised to PhA- after dermal exposure slower and to a lower extent than after oral exposure, which is explained by an extensive first-pass metabolism in the liver. The slower and lower metabolisation of PhE with the dermal route is explaining the qualitatively different toxic effects (haematotoxicity) observed by action of the unmetabolised PhE after dermal application (compared to the oral route), especially in the rabbit.

Analogue approach for the oral route
For more details see the attached justification.
A NOAEL of 400 mg PhE / kg bw (taken from the NTP multi-generation reproduction toxicity test) can be used to read-across to the worst case NOAEL of 408 mg NaPhA / kg bw.
The overall transcription factor is 1.02 if the unit 'mg test substance per kg body weight' is selected.

4. DATA MATRIX
A data matrix is not relevant for this metabolic pathway analogue approach, because the molecule PhE and the anion PhA- are related by metabolism and not by chemical structure or molecular weight or by common physico-chemical properties.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

12 750 mg/kg diet

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Effect level:

12 750 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Dose descriptor:

NOAEL

Effect level:

2 550 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

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 550 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

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

2 550 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

12 750 mg/kg diet

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

yes

Executive summary:

A read-across to a multigeneration study with 2-phenoxyethanol (PhE) in mice is performed. PhE is the metabolic precursor of phenoxyacetate, which is the common anion to the target substance sodium phenoxyacetate and also to phenoxyacetic acid. A read-across analogue approach is described and attached to the target record.

In summary the reproductive toxicity of PhE was only evident in the female and occurred at doses which elicited general toxicity. 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, for females a higher NOAEL corresponding daily intake was calculated.

The transcription of the NOAEL from PhE to NaPhA according to the Analogue Approach provides a NOAEL(NaPhA) = 408 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

408 mg/kg bw/day

Study duration:

subchronic

Species:

mouse

Quality of whole database:

Reliability 2.

Additional information

Justification for selection of Effect on fertility via oral route:
The oral route is suitable to derive dose descriptors for the inhalation and the dermal route. See the appropriate waivers for the repeated dose toxicity endpoints, and also the description and justification for the analogue approach in Section 7.8.1.
Justification for selection of Effect on fertility via inhalation route:

The oral route is suitable to derive dose descriptors for the inhalation and the dermal route. See the appropriate waivers for the repeated dose toxicity endpoints, and also the description and justification for the analogue approach in Section 7.8.1.

Justification for selection of Effect on fertility via dermal route:

The oral route is suitable to derive dose descriptors for the inhalation and the dermal route. See the appropriate waivers for the repeated dose toxicity endpoints, and also the description and justification for the analogue approach in Section 7.8.1.

Effects on developmental toxicity

Description of key information

A weight of evidence approach, including two analogue approaches, was performed.

One source study is an oral developmental toxicity study with mice using phenoxyacetic acid. Phenoxyacetic acid (PhAA) and the target molecule sodium phenoxy acetate (NaPhA) have the same common anion phenoxy acetate in aqueous medium at pH > ca. 4. This study is an old one, not meeting the procedures described in recent guidelines. No significant embryotoxic, foetotoxic and teratogenic effects were observed. No NOAEL, comparable to NOAEL for guideline studies, was obtained.

The other source study was performed with 2-phenoxyethanol (PhE) in rabbits and dermal exposure. The method used was equivalent to present guidelines. PhE is the precursor of the metabolite phenoxyacetate, which is the common anion to the target substance sodium phenoxyacetate and phenoxyacetic acid. A read-across analogue approach is described and attached to the target record.

No significant embryotoxic, foetotoxic and teratogenic effects were observed also in this study.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

<1979

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: The method used does not meet presently accepted procedures. E.g. the dosing scheme.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target salt, i.e. sodium phenoxyacetate (NaPhA), and its free acid phenoxyacetic acid (PhAA,; the source substance) have the same anion phenoxyacetate (PhA-) in aqueous media.
A read-across to properties of the free acid phenoxyacetic acid (PhAA) is suitable for the sodium salt (NaPhA), or vice versa, as both substances are water soluble and dissociate in water to the same anion phenoxyacetate (PhA-). Water is abundantly present in most toxicological systems, except for exposure to skin or for a few ecological systems. For the read-across it is assumed that the introduced cations (hydrogen or sodium ions) are toxicologically not relevant in a buffered environment.
The dissociation constant pKa of PhAA is 3.16, indicating a complete dissociation in neutral systems and generally in systems of pH >ca. 4.
The water solubility of both substances is high, confirming the presence of ions in solution:
PhAA: 12 g/L at 10 °C;
NaPhA: 224 g/L at 20 °C.
Conclusion: A read-across of toxicological data from PhAA to NaPhA or vice versa is possible in aqueous environments - like body fluids or inside of cells - because of the common anion PhA-.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
2.1 Sodium phenoxyacetate
Synonyms: Acetic acid, 2-phenoxy-, sodium salt (1:1); etc.
Abbreviated to: NaPhA (or NaPhAA)
CAS-No.: 3598-16-1
Molecular Formula: C8H7NaO3
Molecular mass: 174.13
Smiles Code: c1(OCC(=O)[O-])ccccc1.[Na+]
Physical state: Solid.

2.2 Phenoxyacetic acid
Abbreviated to: PhAA
CAS: 122-59-8
Molecular formula: C8H8O3
Molecular mass: 152.149
SMILES: O=C(O)COc1ccccc1
Physical state: Solid.

NaPhA and PhAA are dissociated in water at pH > ca. 4. Therefore the common anion phenoxyacetate (PhA-) also occurs as an analogue and in addition the cations Na+ and H+.

3. ANALOGUE APPROACH JUSTIFICATION
See above under "Hypothesis". The dose descriptors can be transcribed from PhAA to NaPhA by considering the molecular masses. E.g. NOAL(NaPhA) = (174.13/152.15) x NOAEL(PhAA) = 1.14 x NOAEL(PhAA).

4. DATA MATRIX
Not relevant.

Reason / purpose for cross-reference:

read-across source

Specific details on test material used for the study:

Phenoxyacetic acid (PA) was obtained from Eastman Kodak, Rochester, New York.

Dose descriptor:

NOAEL

Effect level:

973 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No effects.

Remarks on result:

other: The dose refers to a single dose on one of the gestation days.

Dose descriptor:

NOAEL

Effect level:

315 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no effects.

Remarks on result:

other: The dose refers to 3 consecutive doses within the gestation days 7 to 15.

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

A lower foetal body weight was observed only when dosed at gestation Day 15.

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Histopathological examinations.

Dose descriptor:

NOAEL

Effect level:

973 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects

Remarks on result:

other: The dose refers to a single dose on one of the gestation days

Dose descriptor:

NOAEL

Effect level:

315 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects

Remarks on result:

other: The dose refers to 3 consecutive doses within the gestation days 7 to 15.

Abnormalities:

no effects observed

Conclusions:

No significant skeletal, visceral or histopathological defects of the foetuses were observed.

Executive summary:

A read-across to a study with phenoxy acetic acid is performed. Phenoxyacetic acid (and two structurally related compounds) were suspended in a 1:1 solution of honey:water and administered by gavage to pregnant mice on one of gestation days 8-15 (copulation plug day = day 1) or on three consecutive days (7-9, 10-12, or 13-15). Doses were 800-900 mg/kg for single and 250-300 mg/kg/day for multiple treatments. No increased prenatal mortality, and no decreased fetal weight were observed compared to the solvent controls. Low incidences, not gaining significance, of increased cleft palate or other gross malformations were seen in all treatment groups. Significant skeletal, visceral or histopathological defects were not observed.

The mean dose of 850 mg/kg/d of phenoxy acetic acid corresponds to a mean dose of 973 mg/kg/d of sodium phenoxyacetate.

The mean dose of 275 mg/kg/d of phenoxy acetic acid corresponds to a mean dose of 315 mg/kg/d of sodium phenoxyacetate.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

<1979

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: The method used does not meet presently accepted procedures. E.g. the dosing scheme.

Qualifier:

no guideline available

Version / remarks:

No guideline available at the time of performance.

Principles of method if other than guideline:

Phenoxyacetic acid (and two structurally related compounds) were administered by gavage to pregnant mice on one of gestation days 8-15 (copulation plug day = day 1) or on three consecutive days (7-9, 10-12, or 13-15). Doses were 800-900 mg/kg for single and 250-300 mg/kg/day for multiple treatments. Mortality and fetal weight were determined. Skeletal, visceral or histopathological foetal anomalies and malformations were investigated. Only the part on phenoxyacetic acid is recorded here.

GLP compliance:

not specified

Limit test:

yes

Specific details on test material used for the study:

Phenoxyacetic acid (PA) was obtained from Eastman Kodak, Rochester, New York.

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

Randombred CD-1 albino mice were obtained from the Charles River Breeding Laboratory and given Wayne Lab Blox and water ad libitum.

Route of administration:

oral: gavage

Details on exposure:

Phenoxyacetic acid (PA) was suspended in honey and water (1:1). The concentration suspended was calculated on the basis that a 30 gram mouse would be given a volume of 0.25 ml.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Mated females were detected by the presence of copulation plugs, and the plug day was designated day one of gestation.

Duration of treatment / exposure:

One or three consecutive treatments.

Frequency of treatment:

The test substance was administered at a dose of 800-900 mg/kg of body weight on one of days 8-15 of gestation or at a dose of 250-300 mg/kg of body weight on three consecutive gestation days (7-9, 10-12, or 13-15).
A minimal lethal dose of 1000 mg/kg was established in preliminary trials.

Duration of test:

Until gestation Day 18.

Dose / conc.:

850 mg/kg bw/day (nominal)

Remarks:

A single dose was 800 to 900 mg/kg bw/d on one of the Days 8 to 15 of gestation.

Dose / conc.:

275 mg/kg bw/day (nominal)

Remarks:

A dose of 250 to 300 mg/kg bw/d was administered on three consecutive days (Days 7 -9; 10-12; or 13-15) of gestation.

No. of animals per sex per dose:

Eight or more litters were produced per treatment day x dose.

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

On gestation day 18, treated females were killed by cervical dislocation. Uteri were exposed and examined to determine the numbers of live, dead, and resorbed fetuses. Live fetuses were examined for gross external malformations and weighed. Two fetuses were randomly chosen from each litter, dissected, and examined for visceral abnormalities; their skulls were stored in 70% ethanol and then subjected to free hand sectioning and examination for malformations of the brain, and oral and nasal cavities. Additional fetuses from each litter were selected at random and placed in cold buffered 10% formalin for a later histopathological examination. All other fetuses were stored in 70% ethanol and later eviscerated, stained with alizarin red S and examined for skeletal malformations.

Ovaries and uterine content:

On gestation day 18, treated females were killed by cervical dislocation. Uteri were exposed and examined to determine the numbers of live, dead, and resorbed fetuses.

Fetal examinations:

Live fetuses were examined for gross external malformations and weighed. Two fetuses were randomly chosen from each litter, dissected, and examined for visceral abnormalities; their skulls were stored in 70% ethanol and then subjected to free hand sectioning and examination for malformations of the brain, and oral and nasal cavities. Additional fetuses from each litter were selected at random and placed in cold buffered 10% formalin for a later histopathological examination. All other fetuses were stored in 70% ethanol and later eviscerated, stained with alizarin red S and examined for skeletal malformations.

Statistics:

Fetal weights were compared by use of a one-way ANOVA followed by Gabriel's multiple range test. The incidences of deaths and resorptions were compared nonparametrically by the rank sum method of Wilcoxon and Wilcox. Incidences of gross malformations, skeletal malformations, and cleft palates were compared by arcsin transformation of means, followed by a one-way ANOVA and Gabriel's multiple range test, In all cases, statistical analyses were done on a per litter basis.

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Number of abortions:

not specified

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Dose descriptor:

NOAEL

Effect level:

850 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No effects.

Remarks on result:

other: The dose refers to a single dose on one of the gestation days.

Dose descriptor:

NOAEL

Effect level:

275 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no effects.

Remarks on result:

other: The dose refers to 3 consecutive doses within the gestation days 7 to 15.

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

A lower foetal body weight was observed only when dosed at gestation Day 15.

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Histopathological examinations.

Dose descriptor:

NOAEL

Effect level:

850 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects

Remarks on result:

other: The dose refers to a single dose on one of the gestation days

Dose descriptor:

NOAEL

Effect level:

275 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects

Remarks on result:

other: The dose refers to 3 consecutive doses within the gestation days 7 to 15.

Abnormalities:

no effects observed

Conclusions:

No significant skeletal, visceral or histopathological defects of the foetuses were observed.

Executive summary:

Phenoxyacetic acid (and two structurally related compounds) were suspended in a 1:1 solution of honey:water and administered by gavage to pregnant mice on one of gestation days 8-15 (copulation plug day = day 1) or on three consecutive days (7-9, 10-12, or 13-15). Doses were 800-900 mg/kg for single and 250-300 mg/kg/day for multiple treatments. No increased prenatal mortality, and no decreased fetal weight were observed compared to the solvent controls. Low incidences, not gaining significance, of increased cleft palate or other gross malformations were seen in all treatment groups. Significant skeletal, visceral or histopathological defects were not observed.

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

<1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

The sodium phenoxyacetate metabolic pathway analogue approach is presented.
The description of this analogue approach - in a more extensive and structured form - is attached.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
1.1. Analogue Hypothesis
The target substance (to be registered) is sodium phenoxyacetate (NaPhA). The source substance is phenoxyethanol. Phenoxyethanol is the metabolic precursor of phenoxyacetate.
The hypothesis is that systemic toxicity caused by oral dosing of PhE to mammals comprise at least the toxicity that would be obtained after oral dosing of NaPhA. The dose descriptor NOAEL for PhE can be transcribed as a worst case to NaPhA.
For more details see the attached justification.

1.2. Applicability domain (AD) of the analogue approach
The approach is suitable for read-across of systemic effects observed in toxicity studies with mammals. Primarily oral toxicity studies are concerned, 1) because the practically complete absorption of the source and the target substance is known, and 2) because haematotoxicity, caused by the not yet metabolised source substance PhE, was observed especially after dermal exposure to rabbits.
The applicability of the approach to toxicity endpoints using the dermal or the inhalation route needs some caution.

The read-across approach is not suitable for ecotoxicity endpoints, because PhE is readily biodegradable to CO2 in aqueous media and no stable metabolite PhA- is formed in ecosystems as in the case of mammalian systems.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Sodium phenoxyacetate
Synonyms: Acetic acid, 2-phenoxy-, sodium salt (1:1); etc.
Abbreviated to: NaPhA (or NaPhAA)
CAS-No.: 3598-16-1
Molecular Formula: C8H7NaO3
Molecular mass: 174.13
Smiles Code: c1(OCC(=O)[O-])ccccc1.[Na+]
Physical state: Solid.

Phenoxyacetic acid
Abbreviated to: PhAA
CAS: 122-59-8
Molecular formula: C8H8O3
Molecular mass: 152.149
SMILES: O=C(O)COc1ccccc1
Physical state: Solid.

NaPhA and PhAA are dissociated in water at pH > ca. 4. Therefore the common anion phenoxyacetate (PhA-) and the cations Na+ and H+ also occur.

Phenoxyethanol
Synonyms: 2-phenoxyethanol; ethyl glycol monophenol ether; ethylene glycol monophenyl ether; phenoxytol; 1-hydroxy-2-phenoxyethane; (2-hydroxyethoxy) benzene
Abbreviated to: PhE; (PE)
CAS-No.: 122-99-6
Molecular Formula: C8-H10-O2
Molecular mass: 138.165
Smiles Code: c1(ccccc1)OCCO
Physical form: Oily, slightly viscous liquid at room temperature

2.1. Purity / Impurities
- NaPhA: The registered substance has a purity >96 %. Impurities are sodium chloride and water. Purity is therefore not a relevant factor for the analogue approach, to provide data for systemic toxicity endpoints only.
- PhAA: Purity is not relevant, as PhA- is the major metabolite of PhE and no studies will be proposed with PhAA. Purity is therefore not a relevant factor for the analogue approach.
- PhE: The analytical purity of the test substances used for the key studies on repeated dose toxicity and developmental toxicity were 99.9 % or above. The purity is considered high enough to not impede the analogue approach.

3. ANALOGUE APPROACH JUSTIFICATION
Supporting physical-chemical properties, toxicokinetics and metabolism of the analogues
For more details see the attached justification.
Conclusion:
Phenoxyacetic acid and sodium phenoxyacetate are easily and practically completely absorbed after oral administration. The common anion phenoxyacetate is excreted practically completely and unchanged in urine.
Phenoxyethanol PhE is rapidly and rather completely absorbed after oral dosing. PhE is rapidly metabolised predominantly to PhA- and rapidly excreted mainly as unchanged PhA-. More than 90 % of an oral dose of 2-phenoxyethanol was excreted in the urine of rats as phenoxyacetic acid within 24 h.
PhE is well absorbed through the skin, but not completely. Remarkable is that PhE is metabolised to PhA- after dermal exposure slower and to a lower extent than after oral exposure, which is explained by an extensive first-pass metabolism in the liver. The slower and lower metabolisation of PhE with the dermal route is explaining the qualitatively different toxic effects (haematotoxicity) observed by action of the unmetabolised PhE after dermal application (compared to the oral route), especially in the rabbit.

Analogue approach for the oral route
For more details see the attached justification.
A dermal NOAEL of 600 mg PhE / kg bw (taken from the developmental toxicity study of Scortichini 1987) can be used to read-across to the worst case dermal NOAEL of 426 mg NaPhA / kg bw.
The overall transcription factor is 0.71, if the unit 'mg test substance per kg body weight' is selected.

4. DATA MATRIX
A data matrix is not relevant for this metabolic pathway analogue approach, because the molecule PhE and the anion PhA- are related by metabolism and not by chemical structure or molecular weight or by common physico-chemical properties.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Principles of method if other than guideline:

2-Phenoxyethanol was applied to the clipped skin of pregnant rabbits on Days 6 through 18 of gestation in order to assess the fetotoxic and teratogenic potential by the dermal route. Rabbits were treated with 0, 300, 600, or 1000 mg/kg/day of 2-phenoxyethanol, and fetuses were examined for external, visceral, and skeletal alterations.

Specific details on test material used for the study:

2-Phenoxyethanol was obtained from Nipa Laboratories, Ltd., Great Britain. Analysis by gas chromatography indicated a purity of greater than 99%.

Species:

rabbit

Strain:

New Zealand White

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Dose of 2-phenoxyethanol.

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

Dose of 2-phenoxyethanol.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Dose of 2-phenoxyethanol.

Mortality:

mortality observed, treatment-related

Description (incidence):

During the course of the study, five rabbits in the 600 mg/kg/day dose group and nine rabbits in the 1000 mg/kg/day dose group died or were sacrificed in extremis. Most deaths occurred between gestation Days 11 and 18 (6 to 13 doses).
Due to the excessive lethality seen at the 1000 mg/kg/day dose level, the remaining animals in this dose group were terminated for humane reasons with no further observations. Due to staggered initiation of treatment, a total of five rabbits at 1000 mg/kg/day had completed the dosing regimen and had been sacrificed on Day 28 of gestation, prior to the decision to terminate the dose group.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In those moribund animals for which hematology parameters were evaluated, RBC counts and PCV values were severely depressed, whereas reticulocytes were elevated (Table 1). In addition, red blood cell fragility was increased (data not shown). The collective interpretation of these data indicated the presence of a regenerative hemolytic anemia in these rabbits.
No other significant hematologic findings except for those due to intravascular hemolysis were observed.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The dark urine observed at gross necropsy was interpreted to be due to hemoglobinuria, as there were no intact red blood cells in the urine sediment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The salient pathologic finding in most of the dead animals was the presence of dark-colored urine in the bladder. In addition to the dark urine, the animals were jaundiced and the kidneys were darkened in color. The gross necropsy findings were those typically observed to be associated with an intravascular hemolytic episode.
The specific cause of death for three animals, two at the 600 mg/ kg/day dose level and one at 1000 mg/kg/ day, could not be determined upon gross necropsy. There were no indications of intravascular hemolysis detected in these animals.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Dose descriptor:

LOAEL

Effect level:

426 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

haematology

mortality

Remarks on result:

other: The effect level was transcribed from PhE to NaPhA.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No adverse effects on fetal body measurements were observed among rabbits dosed dermally with 300 or 600 mg/kg/day of 2-phenoxyethanol.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In the control group, single fetuses from different litters exhibited microphthalmia and anonychia.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Alterations of the skeletal system occurred with similar frequency in the control and treated groups. One control fetus exhibited oligodactyly (missing digit), and one fetus in each of the two treated groups had clinodactyly (lateral deflection of a digit). One fetus in the 600 mg/kg/day dose group had a hemivertebra.

Visceral malformations:

no effects observed

Description (incidence and severity):

No malformations of the internal organs were seen in the control or treated groups.

Other effects:

no effects observed

Description (incidence and severity):

crown-rump length.

Dose descriptor:

NOAEL

Effect level:

426 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed.

Remarks on result:

other: The effect level was transcribed from PhE to NaPhA.

Abnormalities:

effects observed, non-treatment-related

Localisation:

other: Single abnormalities and malformations scattered over the control and the treatment groups.

Description (incidence and severity):

In the control group, single fetuses from different litters exhibited microphthalmia and anonychia. Alterations of the skeletal system occurred with similar frequency in the control and treated groups. One control fetus exhibited oligodactyly (missing digit), and one fetus in each of the two treated groups had clinodactyly (lateral deflection of a digit). One fetus in the 600 mg/kg/day dose group had a hemivertebra. No malformations of the internal organs were seen in the control or treated groups.

Developmental effects observed:

yes

Lowest effective dose / conc.:

710 mg/kg bw/day (nominal)

Treatment related:

no

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

no

Conclusions:

Examination of rabbit fetuses indicated that, at the dosages tested, 2-phenoxyethanol was not embryotoxic, fetotoxic, or teratogenic.

Executive summary:

A read-across from a dermal developmental toxicity study with rabbits using 2-phenoxyethanol (PhE) to sodium phenoxyacetate (NaPhA) was performed applying an analogue approach.

2-Phenoxyethanol was applied to the clipped skin of pregnant rabbits on Days 6 through 18 of gestation in order to assess the fetotoxic and teratogenic potential by the dermal route. Rabbits were treated with 0, 300, 600, or 1000 mg/kg/day of 2-phenoxyethanol, and fetuses were examined for external, visceral, and skeletal alterations. Dermal application of 1000 mg/kg/day produced maternal toxicity as evidenced by intravascular hemolysis of red blood cells and death in some animals. Maternal toxicity (intravascular hemolytic episode) was observed in rabbits treated with 600 and 1000 mg PhE/kg/day. Nine rabbits in the 1000 mg/kg/day dose group and five rabbits at 600 mg/kg/day died or were sacrificed in extremis. This haematotoxicity is a known effect of the not yet metabolised PhE and is not of relevance for NaPhA, see the Analogue Approach, attached.

Rabbits in the two highest dose groups which survived until Day 28 of gestation showed no evidence of treatment-related effects. No signs of maternal toxicity were also seen at 300 mg/kg/day.

Examination of rabbit fetuses indicated that, at the dosages tested, 2-phenoxyethanol was not embryotoxic, fetotoxic, or teratogenic.

The NOAEL for foetal toxicity is 426 mg NaPhA/kg bw/d. The NOAEL for maternal toxicity is lower, due to haematotoxicity, which is not relevant for NaPhA.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

<1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Principles of method if other than guideline:

2-Phenoxyethanol was applied to the clipped skin of pregnant rabbits on Days 6 through 18 of gestation in order to assess the fetotoxic and teratogenic potential by the dermal route. Rabbits were treated with 0, 300, 600, or 1000 mg/kg/day of 2-phenoxyethanol, and fetuses were examined for external, visceral, and skeletal alterations.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

2-Phenoxyethanol was obtained from Nipa Laboratories, Ltd., Great Britain. Analysis by gas chromatography indicated a purity of greater than 99%.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Male and female New Zealand White rabbits were obtained and allowed to acclimate to laboratory conditions for at least 2 weeks prior to breeding.
The rabbit was chosen as the test species based on the studies of Hanley et al. (1984), where the rabbit was shown to be more sensitive to ethylene glycol monomethyl ether than either rats or mice.
Female rabbits, approximately 3.5 to 4.5 kg, were used. Animals were housed singly in wire-bottomed cages in rooms designed to control temperature at approximately 22°C and relative humidity at 50% and with a 12-hr light/dark photocycle.
Feed and municipal tap water were available ad libitum.
All animals in the study were uniquely identified using numbered metal eartags.

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

Groups of 25 inseminated rabbits were treated with 300, 600, or 1000 mg/kg/day of undiluted 2-phenoxyethanol, applied dermally, on Days 6 through 18 of gestation. The dose volume of undiluted 2-phenoxyethanol (density = 1.1) was 0.27, 0.55, and 0.91 ml/kg for the 300, 600, and 1000 mg/kg/day dose levels, respectively. Selection of dose levels was based on results of a previous study in which groups of 10 animals were treated with 0, 300, 600, or 1000 mg/kg/day of 2-phenoxyethanol on Days 6 through 18 of gestation. Minimal maternal toxicity as evidenced by a lower body weight gain on Days 15 through 18 of gestation was seen at 1000 mg/kg/day. The highest dose level of 1000 mg/kg/day was chosen due to the physical constraints, i.e., application of a liquid to a finite area of the animal's back without excessive runoff at the time of application or subsequent loss into the occluding bandage.

Prior to insemination on Day 0 of gestation, a section on the back of each rabbit was clipped with electric clippers. Test material was applied to the clipped area daily, beginning on Day 6 of gestation. The application site was occluded using a piece of absorbent gauze and nonabsorbent cotton covered by a cotton flannel bandage held in place with tape. The bandages remained in place 24 hr a day throughout the treatment period (Days 6 through 18 of gestation). Prior to each application of test material, the skin at the application site was examined for signs of irritation and regrowth of hair. Bandages were replaced and the application site clipped as needed during the treatment period. On Day 19 of gestation, the bandages were removed and the application site washed with water to remove any residue of test material to prevent oral ingestion.

Details on mating procedure:

Female rabbits were artificially inseminated, with the day of artificial insemination considered as Day 0 of gestation. Randomization of test animals, grouped according to their Day 0 of gestation, was performed using computer-generated tables of random numbers.

Duration of treatment / exposure:

Inseminated rabbits were treated on Days 6 through 18 of gestation.

Frequency of treatment:

Once a day

Duration of test:

Until gestation Day 28.

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Groups of 25 inseminated rabbits were treated with 300, 600, or 1000 mg/kg/day of undiluted 2-phenoxyethanol, applied dermally, on Days 6 through 18 of gestation. The dose volume of undiluted 2-phenoxyethanol (density = 1.1) was 0.27, 0.55, and 0.91 ml/kg for the 300, 600, and 1000 mg/kg/day dose levels, respectively. Selection of dose levels was based on results of a previous study in which groups of 10 animals were treated with 0, 300, 600, or 1000 mg/kg/day of 2-phenoxyethanol on Days 6 through 18 of gestation. Minimal maternal toxicity as evidenced by a lower body weight gain on Days 15 through 18 of gestation was seen at 1000 mg/kg/day. The highest dose level of 1000 mg/kg/day was chosen due to the physical constraints, i.e., application of a liquid to a finite area of the animal's back without excessive runoff at the time of application or subsequent loss into the occluding bandage.

Control animals:

yes

yes, sham-exposed

Maternal examinations:

Animals were observed daily for evidence of treatment-related effects.
Maternal body weights were recorded on gestation Days 6 through 19 and on Day 28.
Blood was collected from an ear vein from approximately 10 animals per dose group (0, 300, and 600 mg/kg/day) on Day 19 of gestation, and 3 animals (2 at 600 and 1 at 1000 mg/kg/day) sacrificed in extremis for the following measurements: packed cell volume (PCV), hemoglobin (Hgb), erythrocyte count (RBC), total leukocyte count (WBC), red blood cell indices (MCV, MCH, MCHC), platelet count, reticulocyte count, osmotic red cell fragility, and WBC differential counts.
Urine was collected at the time of necropsy from the bladders of two moribund rabbits (one each at 600 and 1000 mg/kg/day) via aspiration for urinalysis (color, appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, blood, urobilinogen, white blood cells, red blood cells, and microscopic examination for crystals and epithelial cells).
Maternal liver weights were recorded at the time of cesarean section on Day 28 of gestation.

Ovaries and uterine content:

The uterine horns were exteriorized through a midline incision in the abdominal wall following carbon dioxide euthanasia, and the number of corpora lutea and the number and position of implantations, resorptions, and live or dead fetuses were recorded. The uteri of apparently non pregnant animals were stained with a 10% solution of sodium sulfide and examined for evidence of early implantation sites. This procedure was performed solely to determine the incidence of pregnancy and was not used to evaluate the incidence of resorptions.

Fetal examinations:

All fetuses were weighed, measured ( crown-rump length), sexed, and examined for external alterations.
One-half of each litter, selected using a table of random numbers, were examined under a dissecting stereomicroscope for evidence of visceral alterations. All fetuses were then preserved in 95% ethanol, cleared, stained with alizarin red S, and examined for skeletal alterations.

Statistics:

Analyses of maternal and fetal body weights, absolute and relative organ weights, appropriate hematologic parameters, and fetal lengths were performed using a parametric or nonparametric analysis of variance followed by either a Dunnett's test or the Wilcoxon rank sum test with Bonferroni's correction. Evaluation of the frequency of preimplantation loss, resorptions among litters and the fetal population, and fetal alterations was performed by a censored Wilcoxon test with Bonferroni's correction. Corpora lutea, implants, and litter size were analyzed with a nonparametric analysis of variance followed by the Wilcoxon rank sum test with Bonferroni's correction. Pregnancy rates were analyzed by the Fisher's exact probability test. Fetal sex ratios were analyzed by a binomial distribution test. The nominal level used for statistical evaluation was a = 0.05. Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) is much greater than the cited alpha levels would suggest. Thus, the final interpretation of numerical data considered statistical analyses, along with other factors such as dose-response relationships and whether the results were significant in light of other biological or pathological findings.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Four rabbits in the 600 mg/kg/day dose group and three rabbits at 1000 mg/kg/day were observed to have darkened areas of skin at the application site. Staining in the perineal region and/or the presence of dark-colored urine underneath their cages was noted in several animals in the middle and high dose groups.

Dermal irritation (if dermal study):

effects observed, non-treatment-related

Description (incidence and severity):

Throughout the dosing period, slight to moderate reddening of the skin at the application site was seen in some animals at all treatment levels.

Mortality:

mortality observed, treatment-related

Description (incidence):

During the course of the study, five rabbits in the 600 mg/kg/day dose group and nine rabbits in the 1000 mg/kg/day dose group died or were sacrificed in extremis. Most deaths occurred between gestation Days 11 and 18 (6 to 13 doses).
Due to the excessive lethality seen at the 1000 mg/kg/day dose level, the remaining animals in this dose group were terminated for humane reasons with no further observations. Due to staggered initiation of treatment, a total of five rabbits at 1000 mg/kg/day had completed the dosing regimen and had been sacrificed on Day 28 of gestation, prior to the decision to terminate the dose group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Pregnant rabbits assigned to the 300 mg/ kg/day dose group weighed significantly more than controls prior to the start of the study; a similar trend continued throughout most of the experimental period. No differences in body weight gains or absolute or relative liver weights were seen between control and treated rabbits.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In those moribund animals for which hematology parameters were evaluated, RBC counts and PCV values were severely depressed, whereas reticulocytes were elevated (Table 1). In addition, red blood cell fragility was increased (data not shown). The collective interpretation of these data indicated the presence of a regenerative hemolytic anemia in these rabbits.
No other significant hematologic findings except for those due to intravascular hemolysis were observed.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The dark urine observed at gross necropsy was interpreted to be due to hemoglobinuria, as there were no intact red blood cells in the urine sediment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The salient pathologic finding in most of the dead animals was the presence of dark-colored urine in the bladder. In addition to the dark urine, the animals were jaundiced and the kidneys were darkened in color. The gross necropsy findings were those typically observed to be associated with an intravascular hemolytic episode.
The specific cause of death for three animals, two at the 600 mg/ kg/day dose level and one at 1000 mg/kg/ day, could not be determined upon gross necropsy. There were no indications of intravascular hemolysis detected in these animals.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No adverse effects on implantations resorbed were observed among rabbits dosed dermally with 300 or 600 mg/kg/day of 2-phenoxyethanol.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No adverse effects on implantations resorbed were observed among rabbits dosed dermally with 300 or 600 mg/kg/day of 2-phenoxyethanol.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No adverse effects on pregnancy rate were observed among rabbits dosed dermally with 300 or 600 mg/kg/day of 2-phenoxyethanol.

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No adverse effects on pregnancy rate were observed among rabbits dosed dermally with 300 or 600 mg/kg/day of 2-phenoxyethanol.

Dose descriptor:

LOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

haematology

mortality

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No adverse effects on fetal body measurements were observed among rabbits dosed dermally with 300 or 600 mg/kg/day of 2-phenoxyethanol.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In the control group, single fetuses from different litters exhibited microphthalmia and anonychia.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Alterations of the skeletal system occurred with similar frequency in the control and treated groups. One control fetus exhibited oligodactyly (missing digit), and one fetus in each of the two treated groups had clinodactyly (lateral deflection of a digit). One fetus in the 600 mg/kg/day dose group had a hemivertebra.

Visceral malformations:

no effects observed

Description (incidence and severity):

No malformations of the internal organs were seen in the control or treated groups.

Other effects:

no effects observed

Description (incidence and severity):

crown-rump length.

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed.

Abnormalities:

effects observed, non-treatment-related

Localisation:

other: Single abnormalities and malformations scattered over the control and the treatment groups.

Description (incidence and severity):

In the control group, single fetuses from different litters exhibited microphthalmia and anonychia. Alterations of the skeletal system occurred with similar frequency in the control and treated groups. One control fetus exhibited oligodactyly (missing digit), and one fetus in each of the two treated groups had clinodactyly (lateral deflection of a digit). One fetus in the 600 mg/kg/day dose group had a hemivertebra. No malformations of the internal organs were seen in the control or treated groups.

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

no

Conclusions:

Examination of rabbit fetuses indicated that, at the dosages tested, 2-phenoxyethanol was not embryotoxic, fetotoxic, or teratogenic.

Executive summary:

2-Phenoxyethanol was applied to the clipped skin of pregnant rabbits on Days 6 through 18 of gestation in order to assess the fetotoxic and teratogenic potential by the dermal route. Rabbits were treated with 0, 300, 600, or 1000 mg/kg/day of 2-phenoxyethanol, and fetuses were examined for external, visceral, and skeletal alterations. Dermal application of 1000 mg/kg/day produced maternal toxicity as evidenced by intravascular hemolysis of red blood cells and death in some animals. Maternal toxicity was observed in rabbits treated with 600 mg 2-phenoxyethanol/kg/day but at a lower incidence than that observed at 1000 mg/kg/day. Nine rabbits in the 1000 mg/kg/day dose group and five rabbits at 600 mg/kg/day died or were sacrificed in extremis. Rabbits in the two highest dose groups which survived until Day 28 of gestation showed no evidence of treatment-related effects. No signs of maternal toxicity were seen at 300 mg/kg/day. Examination of rabbit fetuses indicated that, at the dosages tested, 2-phenoxyethanol was not embryotoxic, fetotoxic, or teratogenic.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

426 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Klimisch 2.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

426 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Klimisch 2.

Additional information

Justification for selection of Effect on developmental toxicity: via oral route:

The oral route is suitable to derive dose descriptors for the inhalation. See the appropriate waivers for the repeated dose toxicity endpoints, and also the description and justification for the analogue approach in Section 7.8.2.

Justification for selection of Effect on developmental toxicity: via inhalation route:

The oral or dermal route is suitable to derive dose descriptors for the inhalation route. See the appropriate waivers for the repeated dose toxicity endpoints, and also the description and justification for the analogue approach in Section 7.8.2.

Justification for selection of Effect on developmental toxicity: via dermal route:

The dermal route is a possible human exposure route.

Justification for classification or non-classification

Results are conclusive but not sufficient for classification.