2-methoxyethyl methacrylate

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

subchronic toxicity study with additional assessment of reproduction toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances are both metabolites of the target substance and fast metabolism occurs.
Therefore, read-across from the existing toxicity studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached “Justification for read-across”

3. ANALOGUE APPROACH JUSTIFICATION
see attached “Justification for read-across”

4. DATA MATRIX
see attached “Justification for read-across”

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

multi-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

NTP continuous breeding protocol
Reproductive assessment by continuous breeding design (RACB). Purpose of study was to develop RACB mouse protocol for use with rats using a known toxicant. Full method described in Chapin (1997). In a modification of the standard protocol, rats were cohabited for ~6 weeks, separated for delivery, nursing and weaning of the second litter for the F2 study, then cohabited for another ~9 weeks to produce a further 3 litters for other activities e.g. cross over mating. Cross over mating was used to determine the affected sex.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: drinking water

Details on mating procedure:

Males and females cohabited for approximately 6 weeks (a deviation from the standard protocol) to allow delivery, nursing and weaning of the 2nd litter, then re-co-habited for approximately nine more weeks.
M/F ratio: 1 to 1. F1 pairs cohabited for 1 week.
Length of cohabitation: 6 week period then 9 week period to produce 3 more litters.
Otherwise standard RACB protocol.

Duration of treatment / exposure:

Continuous from 1 week before mating through F1 generation.

Remarks:

0.01, 0.03, and 0.10% in drinking water (litter two)

Remarks:

0.006, 0.012, and 0.024% in drinking water (litter five)

No. of animals per sex per dose:

20 pairs per treatment group, 40 pairs of control animals

Control animals:

yes

Details on study design:

Dosing for 1 week prior to pairing. F1 pups weaned on postnatal day 21 and weighed weeks 21-23. 2nd generation from 2nd litter of F0 animals.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: weeks 1-6, 10, 15, 18 and after every litter. F1 animals at weaning and weeks 31-33.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Calculated based on data from weeks when body weight and consumption measured on a per cage (per breeding pair) weekly average.

Oestrous cyclicity (parental animals):

Yes. Relative frequency of oestrus, proestrus, metestrus, diestrus, cycle length.

Sperm parameters (parental animals):

Parameters examined included motility, density, and percent abnormal. Sperm analysis is contained in a separate appended report to the main reference.

Litter observations:

-Drinking water consumption monitored week 21. Body weights. F2 pups monitored for 21 days (on days 0, 4, 7, 14, 21).
- standardization of litters: No
- parameters examined: litters per pair, live pups/litter, proportion of pups born alive, sex ration, live pup weight, (adjusted and unadjusted.)
- Gross examination of dead pups:
Postmortem examinations (parental animals)
- Sacrifice: Male animals: At the end of the crossover examination. Maternal animals: not examined.
- Gross necropsy: Yes
- histopathology/organ weights: liver, kidneys, right testis and epididymis and cauda epididymis, seminal vesicles and prostate, ovary. Histopathology only on males in the control and two lower dose groups as previous data indicated no effects likely on females. 10 randomly selected animals per dose group per generation examined. Livers only examined if gross effects seen.

Reproductive indices:

Number of litters and live pups per litter, proportion of live pups, sex ratio. Mating index.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights were significantly reduced from week 3 in females and week 6 in males in the high dose group only.

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption declined from week 2 onwards in both sexes in the high dose group. It was significantly reduced in the mid dose group in week 6 only (4 other measurements not significantly different.)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Testicular and epididymal lesions seen, but also present in controls. Effects were not attributed to treatment.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Motility and density were decreased in the top dose group. Motility increased marginally but by a statistically significant margin in the mid dose group. All parameters were normal in the low dose group.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

100% fertility in controls and low dose group, 89% in mid dose group and only 5% (significantly lower – single breeding pair) in the top dose group. Days to litter were similar in the control and low dose group but increased in the mid dose group. (Too few in high dose group.). The number of litters per pair was not affected in the low and mid dose group. But the number of live pups per pair was significantly reduced in the mid dose group.

Dose descriptor:

NOAEL

Effect level:

0.01 other: % in drinking water; corresponding to 11 mg/kg bw/d

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: Adverse effects on sperm and male reproductive organs

Critical effects observed:

yes

Lowest effective dose / conc.:

33 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Few pups born to high dose animals. Live pups/litter and proportion of live pups/litter decreased significantly in the mid dose group. The proportion of still births, the average postnatal survival were significantly decreased in the mid dose group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of mid dose pups were significantly reduced for both sexes at all time points except one. Adjusted live pup weight increased significantly in the low dose group albeit by only around 5%. F1 mature animals showed reduced body weight (-14%) in the mid dose group.

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

effects observed, treatment-related

Description (incidence and severity):

Drinking water consumption decreased in the mid dose group from week 21. The estimated daily dose was 9 and 27 mg/kg for the low and mid dose group male pups and 15/41 mg/kg respectively for females.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was no effect on adjusted organ weight of females. In males, liver, kidney, seminal vesicle, cauda epididymis, right epididymis and prostate all showed lower weights in the mid dose group after adjustment. The low dose group animals were similar to controls

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Only the testes and epididymis were examined. Minimal to mild testicular lesions were seen in 2/10, 7/10, 5/10 animals in the controls/mid/high dose animals respectively. These results suggested a slight effect from treatment but the no clear dose response was evident. Single animals in the mid and high dose animals showed epididymis lesions, which were not attributed to treatment.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

0.01 other: % in drinking water, corresponding to 11 mg/kg bw/d

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: number of live pups per litter and proportion of pups born alive decreased significantly

Critical effects observed:

not specified

There were no differences in epididymal sperm morphology or motility among the groups from the F2 generation but there was significantly decreased sperm density in both dose groups (-17% in low dose group, -23% in mid dose group).

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

0.01 other: % in drinking water, corresponding to 11 mg/kg bw/d

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Reduced number of live pups per litter

Critical effects observed:

not specified

Reproductive effects observed:

yes

Lowest effective dose / conc.:

33 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

"Reproductive Assessment by Continuous Breeding" (RACB) protocol was originally designed using mice as the test species. The purpose of the present study was to develop a RACB protocol in CD Sprague-Dawley rats. Ethylene glycol monomethyl ether (EGMME), a known reproductive toxicant, was used as the test article.

Reproductive Toxicity of Ethylene Glycol Monomethyl Ether in Sprague-Dawley Rats, Litter Two:
Dose levels selected were 0.01, 0.03, and 0.10%, administered via drinking water. In a modification of the standard protocol, male and female rats ~20 pairs per treatment group, 40 pairs of control animals) were cohabited for approximately 6 weeks, separated to allow delivery, nursing and weaning of the second litter, then re- cohabited for approximately nine more weeks. The weaned second litter was used for F1 reproductive testing. The control and 0.03% F0 pairs were also utilized for a crossover mating trial to determine the affected sex.
Only one litter was born in the 0.10% dose group, and no pups were available for F1 testing. At 0.03% level of EGMME, number of live pups per litter and proportion of pups born alive decreased significantly, both in F0 and F1 testing. In the crossover mating, proportion of pups born alive decreased significantly in the 0.03% male X control female group. Declining control F0 fertility and productivity noted in this study indicate that the standard RACB design (weaning last rather than second litter) is better suited to use with rats.

Reproductive Toxicity of Ethylene Glycol Monomethyl Ether in Sprague-Dawley Rats, Litter Five:
Dose levels selected were 0.006, 0.012, and 0.024%, administered via drinking water. Male and female rats (20 pairs per treatment group, 40 pairs of control animals) were continuously exposed for a 7-day precohabitation period and 112-day cohabitation (Task 2).
While there was no decrease in average litter size with increased dose, the number of live male pups per litter and the total number of pups per litter were decreased in the 0.024% group. Both absolute and adjusted live pup weight were increased in all EGMME groups, but this was not dose-related. During the cross-over mating to determine the affected sex, there were fewer live male pups born to the 0.024% male X control female pairs. During the mating trial for the second generation, fewer male and total pups were delivered in the high-dose group, and both absolute and adjusted pup weight were increased in the middle and high dose groups. Low control fertility (63% fertile) during the cross-over mating is a cause for concern, however suggested protocol changes should alleviate some of this problem. Thus the RACB protocol can successfully be adjusted for use in rats.

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

fertility, other

Remarks:

repeated dose toxicity study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Assessment of the effect of the test substance on sperm and fertility of male rabbits. As a secondary experiment, females were artificially inseminated with sperm from treated males to assess viability of sperm.

GLP compliance:

no

Species:

rabbit

Strain:

Dutch

Sex:

male

Route of administration:

oral: drinking water

Details on mating procedure:

animals were not mated

Duration of treatment / exposure:

12 weeks

Frequency of treatment:

5 d/week

Remarks:

0, 12.5, 25, 37.5, 50 mg/kg bw/d

No. of animals per sex per dose:

6 per treatment group. 7 in highest dose group

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes, weekly. Only start and end reported.

FOOD CONSUMPTION AND COMPOUND INTAKE: Not reported

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Monitored by weighing and compound intake calculated to be 99.7% of nominal.

Sperm parameters (parental animals):

Parameters examined: Parameters measured using Hamilton Thorne computer aided sperm analyser: curvilinear velocity (VCL) , progressive velocity (VSL), path velocity (VAP), beat cross frequency (BCF), amplitude of lateral head displacement (ALH), motility (MOT), progressive spermatozoa (PRS) % and total sperm per ejaculate (SPN). Visual checking of some samples was done to confirm accuracy of automated analysis. All dose groups analysed. Sperm was assessed within 30-45mins of collection.

Sperm were removed from the cauda epididymides of each male in replicate group 1 at necropsy and a wet smear (subsequently dried and stained) of 100 sperm on each of two slides per male were examined and classified as either proximal, midpiece and distal droplets, bent or coiled tail, no tail, abnormal head shape or miscellaneous abnormality. In replicate group 2 the sperm was taken in weeks 11/12.

Postmortem examinations (parental animals):

SACRIFICE
-Animals were weighed and a blood sample taken immediately, prior to IV administration of thiamylal sodium.

GROSS PATHOLOGY
-Weighing of kidney, liver, hear, lung, adrenals, and brain. Testes, caput epidymides and cauda epididymides and accessory sex glands were weighed separately.

-HISTOPATHOLOGY
Organs per gross pathology and fixed in buffered formalin. One testes per animal frozen, the other fixed in Zenker-formal solution after cutting tunica albuginea to assure rapid fixative penetration. Tissue sections stained with H&E.

Statistics:

Males and replicates treated as random variables and treatments as fixed variables. Analysis of variance used. Buck weekly means used for each CASA variable to reduce data set size. Variations within individual males were partitioned for a more precise test of the treatment effects. The fertility tests were factorial with all sperm numbers tested using semen from control and treated males. Means were compared using Tukey’s HSD test with p<0.5 for significance.

Clinical signs:

no effects observed

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:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no differences between replicates so results were combined for analysis. The volume of ejaculate was not affected by treatment but there was a notable decline in sperm number. To the extent that not all could be analysed by CASA. At 25mg/kg PRP was significantly affected. At 37.5mg/kg also VCL, VSL, VAP, MOT, PRS and SPN were significantly affected. There was no change evident in ALH or BCF. The numerical results underestimated the effect at higher doses as they exclude the oligospermic or azoospermic animals that produced too few sperm to analyse. At the top dose, only one animal produced analyzable ejaculate after 12 weeks apart from the two outlier animals. Sperm output from the latter had declined 20% compared to controls but this change was not statistically significant. The effects of treatment on the different types of sperm were small. None were evident from the sperm samples taken from the cauda epididymides. A statistically significant increase in the level of sperm with bent or broken tails or other miscellaneous defects was only seen in ejaculated sperm in the 37.5mg/kg dose group. Sample sizes were low.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All males ejaculated at every semen collection with no effect on libido. A total of 2839 unfertilized and fertilized eggs were collected from females during testing to assess sperm viability. Whilst sperm numbers affected fertility as expected, there was no significant difference between the treatment groups, indicating that fertility was not compromised even though sperm number was produced.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Considerable reduction in size of testes weights from 25mg/kg upwards, ultimately showing a 70% reduction compared to controls. 2 of the 6 animals in the top dose showed no weight reduction. These were considered outliers and eliminated from the data analysis. The weights of the caput and cauda epididymides appeared smaller in the top dose group. No other organ weight changes were evident.

GROSS PATHOLOGY (PARENTAL ANIMALS)

HISTOPATHOLOGY (PARENTAL ANIMALS)
From 25 mg/kg upwards, there was evidence of disturbed spermatogenesis. In the highest dose group there was almost complete disruption (except in the two outlier animals.) Leydig cells appeared normal.

OTHER FINDINGS (PARENTAL ANIMALS)
No effect on blood parameters. No cause was identified for the two outliers in the top dose group who remained almost unaffected by treatment.

Dose descriptor:

NOAEL

Effect level:

12.5 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: Reproductive organ weight changes, effect on most measured sperm parameters

Dose descriptor:

LOAEL

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: Reproductive organ weight changes, effect on most measured sperm parameters

Critical effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Reproductive effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Methoxyethanol when administered to male rabbits for 12 weeks via drinking water produced a marked inhibition of normal spermatogenesis. The effect is marked with a NOAEL of 12.5 mg/kg and a LOAEL of 25 mg/kg. The effect is very specific with no clear effects on sperm morphology and no effect on the ability of the sperm that remain to fertilize a female rabbit. No other adverse effects that could be attributed to treatment were observed at the LOAEL.

Cross-reference 3

Reason / purpose for cross-reference:

read-across: supporting information

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

2-methoxyethyl methacrylate

EC Number:

230-241-9

EC Name:

2-methoxyethyl methacrylate

Cas Number:

6976-93-8

Molecular formula:

C7H12O3

IUPAC Name:

2-methoxyethyl 2-methylprop-2-enoate

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: Adverse effects on sperm and male reproductive organs

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

not measured/tested

Target system / organ toxicity (P1)

Critical effects observed:

not specified

Results: F1 generation

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: number of live pups per litter and proportion of pups born alive decreased significantly

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Reduced number of live pups per litter

Target system / organ toxicity (F2)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes