2-phenoxyethyl isobutyrate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed in line with GLP and the method was designed in line with good scientific principles. The study predates the inception of the OECD guideline 416 (2-generation reproductive toxicity), but is in basic compliance with the current standardised guideline. There is a good level of detail in reporting, sufficient to assess the quality of the data. The read-across approach has been used since phenoxyethanol is structurally similar to the test material but is more toxicologically active. The results presented are therefore taken to be the worst case scenario.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

The reproductive toxicity of the test material was investigated in Swiss CD-1 mice. Mice were exposed to the test material in diet at concentrations of 0, 0.25, 1.25 and 2.5% in diet equivalent to 0, 0.4, 2.0 and 4 g/kg bw/day (a range finding study was conducted with concentrations of 0, 1, 2.5, 5, 7.5 and 10% in feed for 2 weeks). Observations during the study included general toxicity, sperm analysis, reproductive performance, pup abnormalities and assessment of possible sex related effects (crossover mating).

GLP compliance:

yes

Limit test:

no

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

IUPAC Name:

2-phenoxyethanol

Test material form:

not specified

Details on test material:

- Name of test material: Ethylene glycol monophenyl ether (EGPE)
- Analytical purity: 94-95 %
- Impurities: Six impurities were identified, none were detected above 1 %

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (P) 11 wks at the beginning of the continuous breeding test; (F1) 74 ± 10 days.
- Housing: Animals were housed by sex in solid bottom polypropylene or polycarbonate cages with stainless steel lids during the pre-mating period. The animals were then housed either individually or in breeding pairs.
- Diet: ad libitum.
- Water: Deionized water available ad libitum.
- Acclimation period: 2 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 °C.
- Photoperiod (hrs dark / hrs light): 14 hours light (10 hours dark).

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: Weekly (diet was shown to lose approximately 5 % over 7 days).
- Mixing appropriate amounts with diet: Each dose level was blended into a small amount of ground diet, which was then added to a pre-weighed portion and mixed.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 98 days as breeding pairs in the continuous breeding phase, in the crossover trial and the offspring fertility assessment animals were cohabited for 7 days
- Proof of pregnancy: vaginal plug
- After successful mating each pregnant female was caged (how): In the offspring fertility test, animals were housed individually after the cohabitation period. During the continuous breeding phase, the animals cohabited for 98 days.
- Any other deviations from standard protocol: In the crossover mating trial, the parental animals from the continuous breeding phase were cross mated with control animals to determine the affected sex. Three groups of 20 pairs were used: control males x control female; high dose males x control females; control males x high dose females. In the offspring fertility check, the last litter produced from the continuous breeding phase and cohabited with a mate from a different litter within the same dose group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No details of the methods used were reported. Aliquots of six representative samples were analysed during the study, the concentration was shown to be within 96 - 105% of the nominal concentration.

Duration of treatment / exposure:

- Range finding test: 14 days.
- Continuous breeding study: 7 day pre-mating dosing followed by 98 days cohabitation.
- In the cross-over mating trial parental animals selected from the continuous breeding study were necropsied after 3 weeks.
- The animals selected for the offspring fertility test were necropsied at the end of the test (after delivery).

Frequency of treatment:

Daily, dosed feed was available ad libitum, apart from the 7 days were breeding pairs were cohabited in the cross-over mating trial.

Details on study schedule:

- F1 parental animals not mated until 74 ± 10 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
4000, 2000, 400 and 0 mg/kg bw /day
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 0.25, 1.25 and 2.5%
Basis:
actual ingested

No. of animals per sex per dose:

- 8 animals/sex/group in each of the range finding dose levels
- 40 breeding pairs in the control group and 20 breeding pairs per dose were using in the continuous breeding phase of the study.
- In the crossover mating test 20 breeding pairs per group (3 in total) were used.
- In the offspring fertility test, 8 to 10 litters from the F1 generation from the control group and the 1.25 % dose level were selected for rearing to sexual maturity. At 74 ± 10 days one to three female and male pups were selected from each surviving litter and selected randomly for breeding. 19 breeding pairs per dose were used in this test.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: A 14-day range finding study was included in order to select an appropriate dose for the study. Five dose groups and a control were exposed to 10, 7.5, 5.0, 2.5, 1.0 and 0.0 % in diet for two weeks. Animals were assessed for clinical signs, bodyweight changes and food consumption. Based on the results of this study, the appropriate doses were selected for the continuous breeding study. As both male and female animals lost 10 % of their bodyweight in the 7.5 and 10 % groups; 0, 0.25, 1.25 and 2.5 % were selected as the dosing levels in the continuous breeding trial.

Examinations

Parental animals: Observations and examinations:

All animals (continuous breeding study, crossover mating trial and the offspring fertility test) were assessed for the following.

CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes calculated as the average g/day/mouse

Sperm parameters (parental animals):

The following parameters examined in all male mice in all tests within the study; sperm count, sperm motility and sperm morphology.

In addition, the following were examined in the continous breeding test and the offspring fertility test (F0 and F1) animals; testis weight and epididymis weight.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring; number and sex of pups, stillbirths, live births, postnatal mortality, weight gain and fertility.

The number of litters produced by the F0 generation in the continuous breeding phase of the study was also evaluated.

GROSS EXAMINATION OF DEAD PUPS: no

Postmortem examinations (parental animals):

ORGAN WEIGHTS
In the male animals from the continuous breeding study, the females from the crossover mating test and both the males and females from the offspring fertility test the liver weights were recorded.

In addition the following organs and tissues from male animals in the continuous breeding test and the offspring fertility test were weighed: right epididymis, right testis, seminal vesicles and prostate.

Postmortem examinations (offspring):

ORGAN WEIGTHS
As listed above, the following organ weights from the pups produced in the continuous breeding study used in the offspring fertility test were examined: liver, right epididymis, right testis, seminal vesicles and prostate.

Statistics:

The findings from the continuous breeding study were assessed using the Cochran-Armitage test to determine whether there was a dose-related trend. In the cross-over mating trial, as it was not possible to test for a dose-related trend, the findings were assessed using a Chi squared test for homogeneity to determine the overall difference in fertility among groups. Fisher’s exact test was As a pairwise comparisons between the control and dosed groups.
Dose groups means for sex ratio and proportion of live pups were assessed using the Kruskal-Wallis test to examine overall difference and Jonckheere’s test to assess ordered differences. Pairwise comparisons of treatment group means were performed using the Wilcoxon-Mann-Whitney U test.

To prevent the number of pups per litter affecting the assessment of average pup weight an analysis of covariance was used, using the average litter size and the covariate including live and dead pups. Least-squares estimated of dose group means, adjusted for litter size were tested using an F test to assess overall equality and a t test to test for pairwise equality. To prevent interference from potential sex differences, these were performed with male and females separately and also both sexes combined.

An analysis of covariance was also used to adjust organ weights appropriately for bodyweight. Unadjusted body and organ weights were analysed by the Kruskal-Wallis and the Wilcoxon-Mann-Whitney U tests. Jonckheere’s test was used to test for dose-related trends.

Reproductive indices:

The following reproductive indices were calculated using equations below:

Mating index (%) = (Number of animals with copulatory plug/Number of animals cohabited breeding pairs) x 100

Fertility index (%) = (Number of fertile pairs/Number of animals with copulatory plug) x 100

Offspring viability indices:

The number of litters and live pups per litter were assessed per fertile breeding pair from this figure the treatment group means were determined. The proportion of live pups was defined as the number of pups born alive divided by the total number of pups produced by each breeding pair. The sex ratio was expressed as the proportion of male pups born alive out of the total number of live pups born to each fertile pair.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Three females died in the continuous breeding study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males only.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males only.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Test substance intake: No treatment related effects.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment related effects.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

2.5 % EGPE in diet reduced the number of litters delivered per breeding pair and reduced litter size and number of live births.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
The test substance was found to have a low general toxicity, with only three female dying in the continuous breeding test.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the continuous breeding study, only a minimal effect was noted on male bodyweight (2 % decrease) with no change on average in females. There was no significant effect on feed consumption. The approximate daily consumption was 5.6 g/day/mouse. At necropsy at the end of the cross-over mating test, there was a significant decrease in the bodyweight for males exposed to 2.5 % EGPE.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
There were no effects on feed consumption. The average daily test substance intake was approximately 400, 200 and 4000 mg/kg bw/day from the 0.25, 1.25 and 2.5 % groups respectively. The test substance intake varied in the females with the stage of gestation due to fluctuations in bodyweight.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
In the Cross-over mating test there were no differences between the treated and control males in respect to sperm concentration, percentage of motile sperm and percentage of abnormal sperm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Continuous exposure in the continuous breeding test did not affect the number of breeding pairs able to produce at least one litter. Exposure to 2.5 % of EGPE was found to reduce the number of litters delivered per breeding pair and significantly reduced the size and proportion of pups born alive. Further assessment of the litters demonstrated that in the high-dose group only 12 out of 20 pairs (60 %) had a fifth litter compared to 36 out of 40 (90 %) in the control. The number of pups per litter was not found to be affected. In the cross-over mating test, the results did not show which sex was affected by administration of the test material. The only difference was live pup weight was significantly decreased by 12 % in the control males crossed with the 2.5 % dosed females.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In the cross-over mating trial, when adjusted for bodyweight, the liver weight was found to be significantly increased in both males and females (21 and 60 % respectively). There were no significant difference in weight of the right testis, prostate and epididymis between controls and the 2.5 % EGPE males.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight, liver weight and number of litters produced, live pup weight and litter size

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

High lethality was seen in the 2.5 % group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Dose related decrease in body weight

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment related effects.

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Exposure to 2.5 % of EGPE in the continuous breeding test was found to reduce the number of litters delivered per breeding pair and significantly reduced the size and proportion of pups born alive. Further assessment of the litters demonstrated that in the high-dose group only 12 out of 20 pairs (60 %) had a fifth litter compared to 36 out of 40 (90 %) in the control. The number of pups per litter was not found to be affected. In the final litters produced by the continuous breeding test, pup lethality was pronounced during the lactation and post-weaning periods in the 1.25 and 2.5 %. By day 21 (weaning) only eight litters in the 1.25 and 2.5 % dosing groups had sufficient pups for the offspring fertility test. From birth to the start of mating, 12 of 87 (14 %) of the selected offspring for the fertility test in the control group, 20 of 113 pups (18 %) in the 0.25 % EGPE group, 33 out of the 84 pups (39 %) in the 1.25 % EGPE dose group and 66 out of the 76 pups (87 %) in the 2.5 % EGPE treatment group had died. Due to the high lethality in the 2.5 % group (25 of 32 males and 21 of 24 females), the offspring fertility test was performed with the pups from the 1.25 % dosing group.

BODY WEIGHT (OFFSPRING)
There was a significant dose related decrease in adjusted live pup weight during the continuous breeding test. In the cross-over mating test live pup weight was significantly decreased by 12 % in the control males crossed with the 2.5 % dosed females. The F1 pups selected for the offspring fertility test (the last litters produced by the continuous breeding test) were found to have dose-related decreased bodyweights at birth, weaning and at mating (the start of the offspring fertility test). At the end of the offspring fertility test, bodyweights were found to be decreased in both males and females dosed with 1.25 % EGPE.

SEXUAL MATURATION (OFFSPRING)
In the offspring fertility test, there were 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 when compared to the control pairs. Consistently, the live pup weights for the F2 generation were reduced. The offspring fertility test did not indicate any effects on sperm concentration, percentage of motile sperm or percentage of abnormal sperm.

ORGAN WEIGHTS (OFFSPRING)
The F1 pups used in the offspring fertility test were found to have increased liver weights when adjusted for bodyweight at the end of the test. There was no effect on right testis, prostate or epididymal weight. Seminal vesicle weight was found to be significantly decreased compared to controls.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight, liver weight and live pup weight

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Reproductive performance

Parameter	Continuous Breeding Test				Crossover Mating Test			Offspring Fertility Test
	Control	0.25% EGPE in diet	1.25% EGPE in diet	2.5% EGPE in diet	Control male x Control female	2.5 % treated male x control female	Control male x 2.5 % treated female	Control	1.25% EGPE in diet
No. with copulatory plugs/No. cohabited					18/18	18/20	14/17	18/19	19/19
Mating index (%)					100	90	82	95	100
No. fertile/No. with copulatory plugs	38/38	20/20	19/20	18/18	16/18	16/18	12/14	17/18	18/19
Fertility index (%)					88	88	86	94	95
Litters per breeding pair^a	4.8 ± 0.1	4.8 ± 0.1	5.0 ± 0.1	4.5 ± 0.2
Live pups per litter^a	11.0 ± 0.4	11.6 ± 0.5	11.3 ± 0.5	9.0 ± 0.8*	9.06 ± 1.01 (16)	9.75 ± 0.90 (16)	7.42 ± 1.03 (12)	11.1 ± 0.5 (19)	10.7 ± 0.4 (19)
Proportion of pups born alive^a	0.98 ± 0.01	0.99 ± 0.01	0.98 ± 0.02	0.90 ± 0.60*	0.86 ± 0.09 (16)	0.96 ± 0.02 (16)	0.92 ± 0.06 (12)	0.98 ± 0.01 (19)	0.99 ± 0.01 (19)
Live pup weight (g)^a	1.65 ± 0.02	1.59 ± 0.03	1.58 ± 0.02	1.52 ± 0.04*
Adjusted live pup weight (g)^ab	1.65 ± 0.02	1.61 ± 0.02	1.59 ± 0.02*	1.48 ± 0.03*	1.71 ± 0.04 (14)	1.72 ± 0.04 (16)	1.51 ± 0.04 (12)*	1.55 ± 0.02 (19)	1.45 ± 0.02 (19)*
* Significantly different from control group (P < 0.05) ^aMean ± SE. Number of observations included in parentheses were appropriate ^bMeans adjusted for total number of live and dead pups per litter by analysis of covariance

 

Table 2: Bodyweights, organ weights and sperm parameters of male mice in the continuous breeding study and male mice in the offspring fertility test

Observation^a,b	Continuous Breeding Study		Offspring Fertility Test
	Control	2.5 % EGPE in feed	Control	1.25% EGPE in feed
Number of males	37	20	20	20
Bodyweight (g)	38.81 ± 0.59	36.63 ± 0.58*	34.61 ± 0.73	30.71 ± 0.59*
Organ weights
Liver (g)	2.05 ± 0.03	2.48 ± 0.05*	1.85 ± 0.04	2.05 ± 0.04*
Right epididymis (mg)	64 ± 4	54 ± 6	50 ± 1	43 ± 1
Right testis (mg)	135 ± 3	139 ± 5	124 ± 4	117 ± 4
Seminal vesicles (mg)	470 ± 15	446 ± 21	357 ± 15	306 ± 14*
Prostate (mg)	49 ± 3	45 ± 4	30 ± 4	33 ± 4
Sperm parameters
Sperm concentration (10^6sperm/g caudal tissue)	633 ± 34	668 ± 32	1054 ± 52	1041 ± 60
Motile sperm (%)	65 ± 4	69 ± 5	55 ± 3	60 ± 1
Abnormal sperm (%)	2.1 ± 0.2	2.6 ± 0.3	4.5 ± 0.4	5.9 ± 1.3
^aMean ±SE ^bOrgan weights adjusted for bodyweight by analysis of covariance * Significantly different from control (P < 0.05)

 

Table 3: Bodyweights and liver weights of female mice from the cross mating study and the offspring fertility test.

Observation^a,b	Cross Mating Study		Offspring Fertility Test
	Control	2.5 % EGPE in feed	Control	1.25% EGPE in feed
Number of females	38	17	18	16
Bodyweight (g)	37.1 ± 0.07	36.1 ± 0.9	30.97 ± 0.52	28.91 ± 0.50*
Liver (g)	2.15 ± 0.07	3.44 ± 0.11*	1.81 ± 0.04	2.08 ± 0.05*
^aMean ±SE ^bOrgan weights adjusted for bodyweight by analysis of covariance * Significantly different from control (P < 0.05)

 

Table 4: Bodyweights of the offspring from the continuous breeding test (litter 5) at birth, weaning and the beginning of the offspring fertility test

Age (days)	Continuous Breeding Test
	Control	0.25% EGPE in diet	1.25% EGPE in diet	2.5% EGPE in diet
Birth (Day 0)^a
Male	1.77 ± 0.03 (34)	1.70 ± 0.04 (15)	1.69 ± 0.07 (18)	1.60 ± 0.04 (12)*
Female	1.68 ± 0.03 (34)	1.66 ± 0.05 (15)	1.66 ± 0.07 (18)	1.52 ± 0.05 (12)*
Weaning (Day 21)^b
Male	9.82 ± 0.44 (41)	9.43 ± 0.45 (42)	7.35 ± 0.27 (33)*	5.84 ± 0.22 (32)*
Female	9.80 ± 0.41 (39)	8.34 ± 0.32 (54)	7.56 ± 0.29 (32)*	5.57 ± 0.24 (24)*
Mating trial (Day 74 ± 10)^c
Male	35.57 ± 0.87 (20)	34.77 ± 0.70 (20)	30.82 ± 0.62 (20)*	28.76 ± 1.94 (3)*
Female	28.00 ± 0.62 (20)	27.15 ± 0.60 (20)	25.69 ± 0.37 (20)*	24.24 ± 0.82 (3)*
* Significantly different from control group (P < 0.05) ^aMean ± SE for male or female pup weight per litter; number of litters evaluated indicated in parentheses ^bMean ± SE for individual pup weights; number of pups weighed indicated in parentheses ^cMean ± SE for individual mice

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, administration of the test material did not affect the ability to produce 5 consecutive litters in the continuous breeding phase of the study; there was a small decrease in the number of pups/litter and in pup weight in the high dose group, 2.5%. When examined in a crossover study, the affected reproductive component was considered to be from the female, however this was considered inconclusive after assessment, with the only finding being reduced pup weight. Fertility was only minimally compromised, however neonatal toxicity was observed, with increased lethality observed throughout lactation, weaning and puberty. Second generation fertility was found not to be affected, a small decrease in live pup weight was observed. The test material was found to be toxic to immature mice of both sexes. Under the conditions of the test, the NOAEL for both parental and reproductive toxicity was found to be 400 mg/kg bw/day.

Executive summary:

The reproductive toxicity of the test material was investigated in Swiss CD-1 mice. Mice were exposed to the test material in diet at concentrations of 0, 0.25, 1.25 and 2.5% equivalent to 0, 0.4, 2.0 and 4 g/kg bw/day (a range finding study was conducted with concentrations of 0, 1.25 and 2.5, 5, 7.5 and 10% in feed for 2 weeks). Observations during the study included general toxicity, sperm analysis, reproductive performance, pup abnormalities and assessment of possible sex related effects (crossover mating).

Under the conditions of the test, administration of the test material did not affect the ability to produce 5 consecutive litters in the continuous breeding phase of the study; there was a small decrease in the number of pups/litter and in pup weight in the high dose group, 2.5%. When examined in a crossover study, the affected reproductive component was considered to be from the female, however this was considered inconclusive after assessment, with the only finding being reduced pup weight. Fertility was only minimally compromised, however neonatal toxicity was observed, with increased lethality observed throughout lactation, weaning and puberty. Second generation fertility was found not to be affected, a small decrease in live pup weight was observed. The test material was found to be toxic to immature mice of both sexes. Under the conditions of the test, the NOAEL for both parental and reproductive toxicity was found to be 400 mg/kg bw/day.