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EC number: 221-499-3 | CAS number: 3121-61-7
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
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- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Effects on fertility
Description of key information
Toxicity to reproduction (OECD 422, oral, rat):
NOAEL (effects on fertility) < 40 mg/kg bw/day;
NOAEL (developmental toxicity) < 40 mg/kg bw/day;
NOAEL (systemic toxicity) < 40 mg/kg bw/day
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 03 Jan - 23 Mar 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP - Guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Food and Consumer Product Safety Authority (VWA), GG Utrecht, The Netherlands
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl:WI(Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: (P) 11 wks
- Weight at study initiation: (P) Males: 255-301 g; Females: 179-205 g
- Housing: during pre-mating, animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). During mating, females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). After mating, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During lactation, pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams, the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. Generally, sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.0-23.7
- Humidity (%): 33-95
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 23 Jan 2012 To: 23 Mar 2012 - Route of administration:
- oral: gavage
- Vehicle:
- propylene glycol
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: test substance formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and test substance. No correction was made for purity of the test substance. Formulations were stored at room temperature (< 35 °C) in the dark.
VEHICLE
- Justification for use and choice of vehicle (if other than water): based on solubility
- Concentration in vehicle: 7.72, 19.3 and 29% for dose levels of 40, 100 and 250/150 mg/kg bw/day, respectively
- Amount of vehicle (if gavage): 5 mL/kg bw - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually in Macrolon plastic cages (MIII type, height 18 cm). - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The dose formulations at target concentrations of 7.72, 19.3 and 29% were analysed for analytical concentrations using HPLC and UV detection at 205 nm. Homogeneity and stability were assessed in the highest and lowest dose formulation (7.72 and 29%) using the same analytical method (HPLC/UV). The analytical concentrations determined in all dose formulations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The highest and lowest dose formulations were homogenous (i.e. coefficient of variation ≤ 10%). After storage, the highest and lowest dose formulations yielded a relative difference of ≤ 10%. Based on these results, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.
- Duration of treatment / exposure:
- P (Males): 31-35 days, i.e. 2 weeks prior to mating, during mating, and up to termination
P (Females): 42-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation - Frequency of treatment:
- once daily, 7 days/week
- Details on study schedule:
- - Age at mating: ca. 13 weeks
- Remarks:
- Doses / Concentrations:
40, 100 and 250/150 mg/kg bw/day (250 mg/kg bw/day: from Day 1 to 11; 150 mg/kg bw/day: from Day 12 to study termination)
Basis:
actual ingested - No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: dose levels were based on a preliminary range finding study, in which 3 female rats per dose group received the test substance at 100, 200 and 400 mg/kg bw/day. Animals treated with 100 and 200 mg/kg bw/day received the test substance for a period of 10 days, whereas animals of the 400 mg/kg bw/day group were only treated for 2 days. On Day 2 of the study, one animal died and piloerection was observed in one animal at 400 mg/kg bw/day. At necropsy, abnormalities of the stomach, lungs and thymus were found in the animals of this dose group. No mortalities occurred at 100 and 200 mg/kg bw/day. At both dose levels, salivation was observed in all animals during the first days of the study (Day 1-3). No adverse changes in body weights and food consumption were observed and macroscopic examination did not reveal any abnormal findings, except for a reduced thymus size in all animals treated with 200 mg/kg bw/day. Based on the results of this range finding study, dose levels for the main study were 40, 100 and 250 mg/kg bw/day.
- Other: from Day 12 of study onwards, the dose level was lowered to 150 mg/kg bw/day for both sexes due to severe toxicity noted at 250 mg/kg bw/day. - Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for mortality at least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed clinical observations were made daily in all animals, at least immediately after dosing (on the peak period of anticipated effects after dosing). Once prior to start of treatment and at weekly intervals during the treatment period observations were also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded.
BODY WEIGHT: Yes
- Time schedule for examinations: body weights were determined on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4. In order to monitor the health status, animals treated at 250/150 mg/kg bw/day were weighed more often.
FOOD CONSUMPTION: food consumption was determined weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes - Sperm parameters (parental animals):
- Parameters examined in P male parental generations:
testis weight, epididymis weight, other: weight of seminal vesicles including coagulating glands - Litter observations:
- PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight
GROSS EXAMINATION OF DEAD PUPS:
no, for external and internal abnormalities; possible cause of death was determined for pups born or found dead. - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals, after completion of the mating period
- Maternal animals: all surviving animals which delivered, on lactation Days 5-7
Females which did not deliver were sacrificed on post-coitum Days 25-27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 and 2 under “Any other information on materials and methods incl. tables” were prepared for microscopic examination and weighed, respectively. Staging of spermatogenesis in testes was performed. - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring were sacrificed on Days 5-7 of lactation.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: all pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk.
GROSS NECROPSY
- Gross necropsy consisted of external examinations. - Statistics:
- The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. - Reproductive indices:
- Mating index (%) = (number of females mated/number of females paired) x 100
Fertility index (%) = (number of pregnant females/number of females paired) x 100
Conception index (%) = (number of pregnant females/number of females mated) x 100
Gestation index (%) = (number of females bearing live pubs/number of pregnant females) x 100
Duration of gestation = number of days between confirmation of mating and the beginning of parturition - Offspring viability indices:
- Percentage live males at first litter check = (number of live male pups at first litter check/number of live pups at first litter check) x 100
Percentage live females at first litter check = (number of live female pups at first litter check/number of live pups at first litter check) x 100
Percentage of postnatal loss Days 0-4 of lactation = (Number of dead pups on Day 4 of lactation/number of live pups at first litter check) x 100
Viability index = (number of live pups on Day 4 post-partum/number of pups born alive) x 100 - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- 250 mg/kg bw/day: 2 males died on Day 2, 1 male was killed on Day 8; 100 mg/kg bw/day: 1 female killed; detailed clinical signs see “Results of examinations: parental animals”
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- 250 mg/kg bw/day: body weight loss; 250/150 mg/kg bw/day: body weight loss during first 2 weeks of post-coitum and during the last week (f); 100 mg/kg bw/day: body weight loss at end of post-coitum (f)
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- 250 mg/kg bw/day: body weight loss; 250/150 mg/kg bw/day: body weight loss during first 2 weeks of post-coitum and during the last week (f); 100 mg/kg bw/day: body weight loss at end of post-coitum (f)
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- abnormal findings in stomach, testes, epididymides, spleen, liver, uterus, mammary gland, thymus: see “Results of examinations: parental animals”
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- effects observed, treatment-related
- Description (incidence and severity):
- 40 and 250/150 mg/kg bw/day: increase in precoital time (f); 250/150 mg/kg bw/day; decreased fertility and conception indices (only 2 of 9 animals pregnant); 100 and 250 mg/kg bw/day: decrease in number of corpora lutea and implantation sites (f)
- Key result
- Dose descriptor:
- LOAEL
- Remarks:
- fertility
- Effect level:
- 40 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: females: increase in precoital time, increased duration of pregnancy; other: histopathological alterations in testes and epididymides
- Key result
- Dose descriptor:
- LOAEL
- Remarks:
- systemic
- Effect level:
- 40 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: females: levels of MCV and MCH were statistically significantly decreased; males: histopathological alterations in the testes and epididymides
- Dose descriptor:
- NOAEL
- Remarks:
- fertility
- Effect level:
- < 40 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: females: increase in precoital time, increased duration of pregnancy; males: histopathological alterations in testes and epididymides
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- < 40 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: females: levels of MCV and MCH were statistically significantly decreased; males: histopathological alterations in the testes and epididymides
- Dose descriptor:
- NOAEL
- Remarks:
- developmental
- Effect level:
- < 40 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: viability index; postnatal loss
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- 40 mg/kg bw/day: lean and pale appearance
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- 40 mg/kg bw/day: 3/10 litters dead, only 4 litters with some live pubs survived; 100 and 250/150 mg/kg bw/day: no litters
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- 40 mg/kg bw/day: slightly decreased body weights
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- 40 mg/kg bw/day: autolysis in dead animals, blue discolouration of the snout, absence of milk in stomach
- Histopathological findings:
- not examined
- Key result
- Dose descriptor:
- LOAEL
- Remarks:
- developmental
- Generation:
- F1
- Effect level:
- 40 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: viability index; postnatal loss
- Reproductive effects observed:
- not specified
- Conclusions:
- CLP: Repr. Cat. 1B, H360FD
Reference
-Unscheduled deaths: at 250 mg/kg, severe parental toxicity was noted. Toxicity consisted of two male animals found dead on Day 2 of study (no cause of death could be determined) and one male animal that was killed in extremis on Day 8 of study (showed ulcerative inflammation in the stomach with resultant peritonitis). At 100 mg/kg bw/day, one female was killed in extremis on Day 21 post-coitum. Clinical signs in males and females found dead at both dose levels included hunched posture, piloerection, pale and lean appearance.
- Scheduled deaths: treatment-related clinical signs were noted at 250 mg/kg bw/day, and consisted of hunched posture (18 animals 1-5 days), piloerection (1 female 2 days), salivation (3 animals 1 day), and rales (1 female 1 day). These findings disappeared during dosing at 150 mg/kg bw/day. Red vagina or bleeding from the vagina was noted for three females treated at 200 and 250/150 mg/kg bw/day on Days 14, 19 and 23 post-coitum, respectively. This was probably bleeding caused by loss of fetuses and considered treatment-related. These females showed total litter loss or implantation sites only. One female treated at 150 mg/kg bw/day showed hunched posture and piloerection on Days 19 to 21 post-coitum. As this was not noted for the other females at this dose, and recovered during further treatment, it was not considered toxicologically significant.
Incidental findings that were noted included salivation, alopecia, scales and scabs. These findings occurred within the range of background findings to be expected for rats of this age and strain and were thus considered to be of no toxicological relevance.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
At 250 mg/kg bw/day, most male animals and a few female animals showed a body weight loss, which slightly recovered during treatment at 150 mg/kg bw/day. Reduced body weight gains were also noted for males at 100 mg/kg bw/day. At 250/150 mg/kg bw/day, reduced body weight gains were observed during the first two weeks of post-coitum and a body weight loss during the last week. A body weight loss was also noted at the end of postcoitum for females treated with 100 mg/kg bw/day. The reduced body weight gains for females of the 100 mg/kg bw/day dose group during the first two weeks of post-coitum was considered a cause of their pregnancy status (i.e. implantation sites only instead of live fetuses) and not considered toxicologically relevant. No changes in body weights were observed at 40 mg/kg bw/day. Since no litters were born at the 100 and 250/150 mg/kg bw/day, no body weight gain of these animals during lactation could be determined.
Reduced food consumption was noted for the first two weeks of treatment at 250 mg/kg bw/day for both sexes. This recovered during treatment at 150 mg/kg bw/day. At the end of post-coitum, a dose related decrease in food consumption was noted for females treated at 100 mg/kg bw/day (Days 17-20) and 250/150 mg/kg bw/day (Days 14-20) when compared to the concurrent control group. The lower food consumption levels at 100 mg/kg bw/day (Days 7 to 17 post-coitum) and 250/150 mg/kg bw/day (Days 7-14 post-coitum) were considered a cause of their pregnancy status (i.e. implantation sites only or not pregnant), and therefore not considered toxicologically relevant. During lactation, females treated at 40 mg/kg bw/day showed reduced food consumption when compared to the concurrent control animals. However, this was considered due to the reduced number of live pups in these litters. No data on food consumption during lactation was obtained for 100 and 250/150 mg/kg bw/day as no litters were born in these groups. All other statistical significant changes were not considered toxicologically relevant as the values were within normal limits.
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Estrous cyclicity was not determined in females. However, there was evidence of cyclic changes in the reproductive tracts of non-pregnant females treated with 250/150 mg/kg bw/day.
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Staging of spermatogenesis in testes provided evidence of test article related impairment to the spermatogenetic cycle at all dose levels in a dose related manner. The most subtle changes were those of enlarged spermatagonia with finely granular cytoplasm and asynchronous tubules in which normal cell associations were absent. Individual cell necrosis, spermatidic giant cells, and reduced layers of spermatagonia were common observations. At the highest 250/150 mg/kg bw/day dose level there were no recognizable stages. Tubules were often lined by a single layer of cells with uncertain identity (Sertoli cells, primary spermatagonia or both). Mitotic figures were occasionally present and appeared abnormal.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): see Table 3 under “Any other information on results incl. tables”)
Male and female reproduction was affected at all dose levels. Precoital time of females treated at 40 and 250/150 mg/kg bw/day was increased. Two females of the high dose group did not mate in the first pairing period of 15 days. Out of these, one female mated with her second male, whereas the other did not. Fertility and conception indices were decreased at 250/150 mg/kg bw/day, as out of the nine mated females only two were pregnant. In addition, a dose related decrease was noted for number of corpora lutea and implantation sites at 100 and 250/150 mg/kg bw/day. At 40 mg/kg bw/day, an increased duration of gestation was noted.
ORGAN WEIGHTS (PARENTAL ANIMALS)
In males treated at 100 and 250/150 mg/kg bw/day, reduced absolute and/or relative weights of the thymus, testes, prostate, epididymides and seminal vesicles were noted (see Table 4 under “Any other information on results incl. tables”). No toxicologically relevant findings were noted for the females. All other statistically significant changes were considered not to be a sign of toxicity as they occurred in the absence of a treatment-related distribution, were due to the lower terminal body weight or were considered due to the pregnancy/lactation status of the females (i.e. for the liver, adrenals, spleen, ovaries).
GROSS PATHOLOGY (PARENTAL ANIMALS)
Testes of reduced size (with or without flaccidity) were present in 8/10 males at 100 mg/kg bw/day and 8/10 males at 250/150 mg/kg bw/day. Epididymides of reduced size (with or without flaccidity) were present in 7/10 males at 100 mg/kg bw/day and 7/10 males at 250/150 mg/kg bw/day. Fore-stomachs (non-glandular portion) had irregular surfaces in 7/10 males and 5/10 females at 250/150 mg/kg bw/day. This observation was also noted in the glandular stomach of 3/10 males at 250/150 mg/kg bw/day. One male and one female at 100 mg/kg bw/day and two males at 250/150 mg/kg bw/day had reduced thymus size. The male animals that did not survive until planned necropsy showed additionally several abnormalities in the following organs: lungs, stomach, liver, seminal vesicles, spleen, mesenteric and mandibular lymph nodes, lacrimal glands, diaphragm, and body cavities. In addition, beginning autolysis was noted for the two animals that were found dead. Macroscopic examination of the female animal that was killed in extremis revealed findings in the uterus, cervix, spleen, mandibular lymph nodes, upper jaw, and abdominal cavity.
The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy observations included findings in the lungs (foci), liver (reduced in size), kidneys (pelvic dilation), epididymides (nodule), mandibular lymph nodes (enlarged, discolouration), uterus (enlarged, thickened, contains fluid), clitoral glands (discolouration), skin (alopecia), and spleen (nodule, enlarged).
HISTOPATHOLOGY (PARENTAL ANIMALS)
Test article related microscopic observations were present in the stomach, testes, epididymides, spleen, and liver. Secondary effects were present in the uterus, female adrenals, female mammary glands, and thymus (see Table 5 under “Any other information on results incl. tables”).
Abnormal findings in the stomach included inflammation, haemorrhage, hyperkeratosis and hyperplasia of non-glandular epithelium, and degeneration of glandular epithelium. Incidences of pathological findings in the stomach are summarised in Table 5 under “Any other information on results incl. tables”.
In the testes, degeneration of seminiferous tubular epithelium, increased severity of edema, chronic active inflammation, and enlarged amphophilic cells were observed. Incidences of pathological findings in the testes are summarised in Table 5 under “Any other information on results incl. tables”. Staging of spermatogenesis in testes provided evidence of test article-related impairment to the spermatogenetic cycle at all dose levels in a dose related manner. The most subtle changes were those of enlarged spermatagonia with finely granular cytoplasm and asynchronous tubules in which normal cell associations were absent. Individual cell necrosis, spermatidic giant cells, and reduced layers of spermatagonia were common observations. At the 250/150 mg/kg bw/day dose level, there were no recognizable stages. Tubules were lined often by a single layer of cells with uncertain identity (Sertoli cells, primary spermatagonia or both). Mitotic figures were occasionally present and appeared abnormal.
Epididymal observations included degenerate sperm, hypospermia, atrophy and inflammation. Incidences of pathological findings in the epididymides are summarised in Table 5 under “Any other information on results incl. tables”.
In the spleen, reduced (but not dose-related) numbers of hematopoietic cells were noted.
Minimal or slight hepatocellular necrosis was observed in the liver of two males and one female at 250/150 mg/kg bw/day.
Secondary effects of treatment were observed in the uterus, adrenal gland, mammary gland and thymus. In the uterus, implantation sites were observed in 5/5, 6/6, 3/8 and 1/6 females at 0, 40, 100 and 250/150 mg/kg bw/day, respectively. Another three females of the 100 mg/kg bw/day group had placental trophoblasts and necrotic debris in the uterine lumen as evidence of litter loss. There was evidence of cyclic changes in the reproductive tracts of non-pregnant 250/150 mg/kg bw/day females. No pregnancy associated hypertrophy of the cortical adrenal was present in the females of all dose groups. In the mammary gland, observations consisted of reduced incidences and severities of normally expected pregnancy-related hyperplasia of the mammary gland. The incidences of hyperplasia were 5/5, 4/4, 4/5 and 0/5 in the respective 0, 40, 100 and 250/150 mg/kg bw/day dose groups with progressive decreases (3.0, 2.3, 1.5 and 0) in average severity. In the thymus, lymphoid cortical atrophy was present in 1/4 males at 100 mg/kg bw/day and 4/10 males 250/150 mg/kg bw/day. In females, these effects occurred in 1/5, 2/6 and 1/5 females at 40, 100 and 250 mg/kg bw/day, respectively. The unscheduled deaths at 100 and 250 mg/kg bw/day showed marked atrophy of the thymus. In the remaining animals, the incidence of the effect was only minimal or slight.
HAEMATOLOGY:
Several haematological parameters were statistically significantly affected by treatment with the test substance. Decreased levels of haemoglobin, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and platelets were noted for males treated at 250/150 mg/kg bw/day. For females, decreased levels of haemoglobin, mean corpuscular volume (MCV), MCH, MCHC and increased prothrombin time were noted at 100 and 250/150 mg/kg bw/day. In addition, levels of MCV and MCH were also statistically significantly decreased for females treated at 40 mg/kg bw/day.
A clear dose-related effect on development was noted at 40, 100 and 250/150 mg/kg bw/day. Implantation sites were only noted for nine females at 100 mg/kg bw/day and two females at 250/150 mg/kg bw/day. The remaining females were non pregnant or did not mate. Therefore, no pups could be examined for postnatal development. Out of the nine litters at 40 mg/kg bw/day, only six had live pups at first litter check. The number of pups per litter was decreased when compared to the control group. In addition, most of these pups did not survive the first days of lactation; only four litters had some live pups at planned necropsy.
CLINICAL SIGNS (OFFSPRING)
At 40 mg/kg bw/day, lean and pale appearance was seen in the surviving pubs.
BODY WEIGHT (OFFSPRING)
Body weights of the pups at 40 mg/kg bw/day were slightly, but not statistically significantly decreased when compared to the control group.
GROSS PATHOLOGY (OFFSPRING)
Macroscopic findings involved absence of milk in the stomach and blue discolouration of the snout. In addition, autolysis was noted for pups found dead.
Table 3. Reproduction data
Parameter |
Dose [mg/kg bw/day] |
|||
0 |
40 |
100 |
250/150 |
|
Mating index [%] |
100 |
100 |
100 |
90 |
No. of females mated |
10/10 |
10/10 |
10/10 |
9/10 |
Fertility index [%] |
100 |
100 |
90 |
20 |
No. of implantation sites# |
11.1 ± 2.0 |
9.8 ± 1.4 |
7.8 ± 4.3 |
3.5 ± 3.5 |
No. of corpea lutea# |
14.5 ± 4.3 |
11.3 ± 1.8 |
9.9 ± 4.5 |
1.1 ± 3.0** |
Duration of gestation [d]# |
21.4 ± 0.5 |
23.1 ± 0.6 |
n.a. |
n.a. |
Conception index [%] |
100 |
100 |
90 |
22.2 |
No. of pregnant females |
10/10 |
10/10 |
9/10 |
2/10 |
No. of non-pregnant females |
0/10 |
0/10 |
1/10 |
8/10 |
No. of females with live pups (Day 1) |
10/10 |
7/10 |
0/10 |
0/10 |
Gestation index [%] |
100 |
70 |
0 |
0 |
Litter size |
10 |
9 |
n.a. |
n.a. |
*/** Steel−test significant at 5% (*) or 1% (**) level, n.a. = not applicable; # mean value ± standard deviation
Table 4. Statistically significant changes in reproduction organ weights of males
Organ weights |
Dose [mg/kg bw/day] |
|||
0 |
40 |
100 |
250/150 |
|
Thymus, absolute [g] |
0.260 ± 0.017 |
0.258 ± 0.033 |
0.141 ± 0.014** |
0.116 ± 0.022** |
Thymus, relative [%] |
0.079 ± 0.006 |
0.083 ± 0.011 |
0.048 ± 0.006** |
0.040 ± 0.007** |
Testis, absolute [g] |
3.31 ± 0.18 |
3.26 ± 0.31 |
1.86 ± 0.30** |
1.46 ± 0.11** |
Testis, relative [%] |
1.02 ± 0.12 |
1.03 ± 0.07 |
0.62 ± 0.10** |
0.51 ± 0.05** |
Epididymides, absolute [g] |
1.133 ± 0.106 |
1.104 ± 0.077 |
0.801 ± 0.091** |
0.645 ± 0.068** |
Epididymides, relative [%] |
0.348 ± 0.043 |
0.349 ± 0.027 |
0.268 ± 0.040** |
0.225 ± 0.027** |
Seminal vesicles, absolute [g] |
1.778 ± 0.222 |
1.460 ± 0.138 |
1.377 ± 0.201* |
1.420 ± 0.213* |
Seminal vesicles, relative [%] |
0.543 ± 0.067 |
0.468 ± 0.049 |
0.471 ± 0.065 |
0.482 ± 0.065 |
Prostate, absolute [g] |
0.662 ± 0.138 |
0.549 ± 0.111 |
0.471 ± 0.075* |
0.413 ± 0.042* |
Prostate, relative [%] |
0.201 ± 0.038 |
0.176 ± 0.035 |
0.161 ± 0.025 |
0.140 ± 0.016* |
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 5. Histopathological findings in reproduction organs/endocrine organs of males and females
Histopathological findings in reproduction organs/endocrine organs |
Dose [mg/kg bw/day] |
|||
0 |
40 |
100 |
250/150 |
|
Number of animals per group |
10 |
10 |
10 |
10 |
Primary effects |
||||
TESTES (males) |
5 |
5 |
9 |
10 |
- Enlarged cells |
- |
- |
- |
2 |
- Degeneration of seminiferous tubular epithelium |
1 |
2 |
9 |
8 |
- Edema |
4 |
3 |
7 |
7 |
- Chronic active inflammation |
- |
- |
- |
2 |
- dilated rete |
- |
1 |
1 |
- |
|
||||
TESTES, PAS STAGING (males) |
5 |
5 |
5 |
8 |
- All stages missing |
- |
- |
1 |
5 |
- Enlarged cells |
- |
- |
- |
2 |
- Most stages missing |
- |
- |
4 |
1 |
- Some stages missing |
- |
- |
- |
1 |
- Multiple acrosomes |
- |
- |
- |
1 |
- Asynchronous tubules |
- |
2 |
4 |
1 |
- Individual cell necrosis |
- |
3 |
3 |
- |
- Reduced spermatagonia |
- |
1 |
5 |
6 |
- Spermatidic giant cells |
- |
1 |
3 |
1 |
- Vacuolation basilar |
- |
- |
1 |
- |
|
||||
EPIDIDYMIDES (males) |
5 |
5 |
8 |
10 |
- Sperm granuloma |
- |
1 |
1 |
- |
- Sperm degeneration |
- |
- |
8 |
8 |
- Hypospermia |
- |
- |
1 |
8 |
- Atrophy |
- |
- |
7 |
8 |
- Chronic active inflammation |
- |
- |
1 |
4 |
Secondary effects |
||||
UTERUS (female) |
5 |
6 |
8 |
6 |
- Stromal hyperplasia |
- |
- |
- |
1 |
- Dilation cyclic |
- |
- |
- |
3 |
- Haemorrhage |
2 |
6 |
3 |
- |
- Inflammation supp |
- |
- |
1 |
- |
- Necrotic debris/neut |
- |
- |
1 |
- |
- Implant sites |
5 |
6 |
3 |
1 |
- Throphoblasts/Necro |
- |
- |
3 |
- |
|
||||
ADRENALS(females) |
5 |
- |
1 |
5 |
- Hypertrophy cortex |
5 |
- |
- |
- |
- Extra cortical nodule |
1 |
- |
- |
- |
- Extramed haematopoies |
- |
- |
1 |
- |
|
|
|
|
|
MAMMARY GLAND AREA(females) |
5 |
4 |
5 |
5 |
- Hyperplasia |
5 |
4 |
4 |
- |
- Inactive gland |
- |
- |
1 |
5 |
- Active gland |
- |
1 |
1 |
- |
- Infiltrate lymphoid |
- |
1 |
- |
- |
|
||||
THYMUS (females) |
5 |
5 |
6 |
5 |
- Increased apoptosis |
- |
1 |
- |
- |
- Haemorrhage/Congestion |
- |
1 |
1 |
- |
- Athrophy lymphoid |
- |
1 |
2 |
1 |
- Hyperplasia duct |
- |
1 |
- |
- |
|
|
|
|
|
THYMUS (males) |
5 |
5 |
4 |
8 |
- Increased apoptosis |
- |
1 |
2 |
- |
- Haemorrhage/Congestion |
- |
1 |
1 |
1 |
- Athrophy lymphoid |
- |
- |
1 |
4 |
Table 6. Developmental data
Parameter |
Dose [mg/kg bw/day] |
|||
0 |
40 |
100 |
250/150 |
|
Pub weight [g]# |
6.0 ± 0.7 |
5.6 ± 0.5 |
n.a. |
n.a. |
Sex ratio [% males] |
42 |
43 |
n.a. |
n.a. |
Viability index [%] |
99 |
66.7** |
n.a. |
n.a. |
Litter size |
10 |
9 |
n.a. |
n.a. |
Dead pubs at first litter check |
|
|||
- Litters affected |
0/10 |
6/10** |
n.a. |
n.a. |
- Total |
0 |
13 |
n.a. |
n.a. |
Living pups at first litter check |
103 |
30 |
n.a. |
n.a. |
- Mean per litter |
10.3 ± 2.4 |
3.3 ± 3.2++ |
n.a. |
n.a. |
Postnatal loss |
|
|
|
|
- Litters affected |
1/10 |
1/9 |
n.a. |
n.a. |
- Total |
1 |
10** |
n.a. |
n.a. |
- % of living pups |
1.0 |
33 |
n.a. |
n.a. |
*/** Fisher's Exact test significant at 5% (*) or 1% (**) level; +/++ Steel−test significant at 5% (+) or 1% (++) level; n.a. = not applicable; # mean values ± standard deviation
Effect on fertility: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- LOAEL
- 40 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
A combined oral repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed with 2 -methoxyethyl acrylate (2 -MEA) according to OECD 422 (de Raaf-Beekhuijzen, 2012). Ten rats per sex and group received the substance at dose levels of 40, 100 and 250/150 mg/kg/day in propylene glycol via oral gavage. Males were treated for a period of 31-15 days (2 weeks prior to mating, during mating, and up to termination), whereas females were administered the test substance for 42-56 days (during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation). Animals of the high dose group were treated with 250 mg/kg bw/day from Day 1 to 11, but this dose was lowered to 150 mg/kg bw/day from Day 12 to study termination due to severe toxicity noted in these animals. One further group of 10 animals per sex received propylene glycol as vehicle and served as controls.
At 250 mg/kg bw/day, severe parental toxicity was noted. Toxicity consisted of two male animals found dead on Day 2 of study and on one male animal that was killed in extremis on Day 8 of study. At 100 mg/kg bw/day, one female was sacrificed in extremis on Day 21 post-coitum. Clinical signs were observed at 250/150 mg/kg bw/day and included hunched posture, piloerection, pale and lean appearance, salivation and rales. Red vagina or bleeding from the vagina was noted for three females treated at 100 and 250/150 mg/kg bw/day on Days 14, 19 and 23 post-coitum, respectively. This was probably bleeding caused by loss of fetuses and considered treatment-related. These females showed total litter loss or implantation sites only. At 250 mg/kg bw/day, a loss in body weight was observed, which slightly recovered during treatment at 150 mg/kg bw/day. Reduced body weight gains were also noted for males at 100 mg/kg bw/day. At 250/150 mg/kg bw/day, reduced body weight gains were observed during the first two weeks of post-coitum and a body weight loss during the last week. A body weight loss was also noted at the end of post-coitum for females treated with 100 mg/kg bw/day. No changes in body weights were observed at 40 mg/kg bw/day.
Male and female reproduction was affected at all dose levels. Precoital time of females treated at 40 and 250/150 mg/kg bw/day was increased. Two females of the high dose group did not mate in the first pairing period of 15 days. Out of these, one female mated with her second male, whereas the other did not. Fertility and conception indices were decreased at 250/150 mg/kg bw/day, as out of the nine mated females only two were pregnant. In addition, a dose related decrease was noted for number of corpora lutea and implantation sites at 100 and 250/150 mg/kg bw/day. At 40 mg/kg bw/day, an increased duration of gestation was noted. Staging of spermatogenesis in testes provided evidence of test article related impairment to the spermatogenetic cycle at all dose levels in a dose related manner. The most subtle changes were those of enlarged spermatagonia with finely granular cytoplasm and asynchronous tubules in which normal cell associations were absent. Individual cell necrosis, spermatidic giant cells, and reduced layers of spermatagonia were common observations. At the highest 250/150 mg/kg bw/day dose level there were no recognizable stages. Tubules were often lined by a single layer of cells with uncertain identity (Sertoli cells, primary spermatagonia or both). Mitotic figures were occasionally present and appeared abnormal.
Absolute and relative organ weights of the thymus, testes, prostate and epididymides were statistically significantly reduced in males treated at 100 and 250/150 mg/kg bw/day. For females at 250/150 and at 100 mg/kg bw/day, absolute and relative liver and ovaries weights were statistically significant reduced.
At all dose levels, test substance related microscopic observations were present in the stomach, testes, epididymides, spleen, and liver. Stomach observations due to corrosive properties of the test substance included inflammation, haemorrhage, hyperkeratosis and hyperplasia of nonglandular epithelium, and degeneration of glandular epithelium. Adverse effects on testes and epididymides were observed at all dose levels. There were reduced but not dose related numbers of hematopoietic cells in the spleen. Hepatocellular necrosis was present in the liver in two males and one female. At 100 mg/kg bw/day, similar findings as at 250/150 mg/kg bw/day were noted, but these occurred at a slightly lower incidence/severity. In contrast to the high dose group, no histopathological effects in liver were found at 100 mg/kg bw/day. Secondary effects were seen in the uterus, female adrenals, female mammary glands, and thymus at all dose levels. Thymic atrophy in a few animals was imputed to be caused by stress associated release of cortical steroids.
In one female treated at 100 mg/kg bw/day, which was sacrificed in extremis showed suppurative inflammation. In offspring, a clear dose-related effect on development was noted at all dose levels. Implantation sites were only noted for nine females at 100 mg/kg bw/day and two females at 250/150 mg/kg bw/day. The remaining females were non pregnant or did not mate. No pubs were born at 100 and 250/150 mg/kg bw/day. Out of the nine litters at 40 mg/kg bw/day, only six had live pups at first litter check. The number of pups per litter was decreased when compared to the control group. In addition, most of these pups did not survive the first days of lactation. At 40 mg/kg bw/day, lean and pale appearance was seen in the surviving pubs and body weights were slightly, but not statistically significantly decreased when compared to the control. Macroscopic findings involved absence of milk in the stomach and blue discolouration of the snout. In addition, autolysis was noted for pups found dead. Based on the results of the study, the NOAEL for systemic, developmental and reproductive toxicity in rats was considered to be lower than 40 mg/kg bw/day.
As 2-MEA showed severe effects on fertility in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (de Raaf-Beekhuijzen, 2012), data on reproductive toxicity meet the criteria for classification as Repr. 1B (H360F) according to Regulation (EC) 1272/2008 and as Repr. Cat. 2 (R60) according to Directive 67/548/EEC.
In addition, there are data available on effects on fertility for the primary metabolite 2-methoxyethanol (CAS no. 109-86-4) which showed similar effects in reproduction toxicity studies as observed for 2-MEA. Based on QSAR estimation using the OECD Toolbox v.2.3, it can be assumed that enzymatic cleavage of the ester bond of 2-MEA results in 2-methoxyethanol and acrylic acid as primary metabolites (for further details on metabolism please refer to the endpoint toxicokinetics).
Studies on 2-methoxyethanol with respect to effects on fertility were consistently toxic to the male reproductive system in multiple species (mice, rats, guinea-pigs, rabbits and dogs) exposed by all routes of administration (subcutaneous, dermal, oral or inhalation) (CICAD, 2009). Effects on reproductive ability as well as reproductive organs have been observed, often at the lowest dose or concentration tested. Single or repeated oral administration of 2-methoxyethanol induced adverse effects on the testes (including weight and histopathological changes or biochemical indicators of testicular damage, such as urinary creatine) and/or various sperm parameters in every identified study in which these endpoints were examined (CICAD, 2009).
With respect to the standard information requirements according to Regulation (EC) 1907/2006 Annex IX, Column I, 8.7.3, a two- or one-generation reproduction toxicity study following OECD 416 or OECD 415 does not need to be conducted if reliable data are available showing adverse effects on fertility according to the criteria for classifying the test substance as R60. Therefore, a GLP-compliant two- or one-generation reproduction toxicity study with 2-MEA is not required. This is also in accordance with Regulation (EC) 1907/2006, which specifies that unnecessary tests should be avoided in terms of animal welfare reasons.
Reference not cited in the IUCLID:
CICAD (Concise Internation Chemical Assessment Document) 67, Selected Alkoxyethanols: 2 -Methoxyethanol, ISBN 92 4 153067 7, WHO, 2009
Effects on developmental toxicity
Description of key information
Short description of key information:
Toxicity to reproduction (OECD 422, oral, rat):
NOAEL (effects on fertility) < 40 mg/kg bw/day;
NOAEL (developmental toxicity) < 40 mg/kg bw/day;
NOAEL (systemic toxicity) < 40 mg/kg bw/day
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- LOAEL
- 40 mg/kg bw/day
- Study duration:
- subacute
- Species:
- mouse
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
A developmental toxicity study evaluating 60 chemicals in mice including 2 -methoxyethyl acrylate (2-MEA) was published by Hardin et al. (1987). 50 pregnant mice were dosed by gavage with 650 mg/kg bw/day of the test substance on gestation days 6 -13. The mice were then permitted to deliver litters. The test substance produced 30% maternal mortality and 100% intrauterine death. Therefore, the test substance adversely affected all measures of reproductive success since no liveborn pubs were recorded. Dead pubs were not examined for malformations. However, it should be pointed out that maternal mortality was 30% and that the dose tested was not suitable for evaluating developmental toxicity.
Moreover, in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (presented under “effects on fertility”; de Raaf-Beekhuijzen, 2012), data on reproductive toxicity indicated that in offspring a clear dose-related effect on development toxicity was noted at all dose levels of 2-MEA. Implantation sites were only noted for nine females at 100 mg/kg bw/day and two females at 250/150 mg/kg bw/day. The remaining females were non pregnant or did not mate. No pubs were born at 100 and 250/150 mg/kg bw/day. Out of the nine litters at 40 mg/kg bw/day, only six had live pups at first litter check. The number of pups per litter was decreased when compared to the control group. In addition, most of these pups did not survive the first days of lactation. At 40 mg/kg bw/day, lean and pale appearance was seen in the surviving pubs and body weights were slightly, but not statistically significantly decreased when compared to the control. Macroscopic findings involved absence of milk in the stomach and blue discolouration of the snout. In addition, autolysis was noted for pups found dead. Based on the results of the study, the NOAEL for developmental toxicity in rats was considered to be lower than 40 mg/kg bw/day.
As 2-MEA caused severe effects on developmental toxicity in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (de Raaf-Beekhuijzen, 2012), data on developmental toxicity meet the criteria for classification as Repr. 1B (H360D) according to Regulation (EC) 1272/2008 and as Repr. Cat. 2 (R61) according to Directive 67/548/EEC.
In addition, there are data available on developmental toxicity for the primary metabolite 2-methoxyethanol (CAS no. 109-86-4) which showed similar effects in developmental toxicity studies as observed for 2-MEA. Based on QSAR estimation using the OECD Toolbox v.2.3, it can be assumed that enzymatic cleavage of the ester bond of 2-MEA results in 2-methoxyethanol and acrylic acid as primary metabolites (for further details on metabolism please refer to the endpoint toxicokinetics).
2-Methoxyethanol has consistently induced developmental toxicity in numerous oral studies in several species of laboratory animals, generally at doses lower than those that are maternally toxic, and often at the lowest exposure level tested (CICAD, 2009). Decreased fetal body weights were noted in rats repeatedly exposed to 2-methoxyethanol doses of 16 mg/kg bw/day or more in the diet during gestation, with malformations being observed at doses of 31 mg/kg bw/day or greater, whereas maternal toxicity was present only at higher doses. Similar results were obtained in several other studies in rats exposed to 2-methoxyethanol in the diet or by gavage. In many of the studies, the cardiovascular system, kidney and skeletal system were the principal targets for 2-methoxyethanol-induced malformations; functional defects of the heart were also noted.
With respect to the standard information requirements according to Regulation (EC) 1907/2006 Annex IX, Column I, 8.7.2., a prenatal developmental toxicity study following OECD 414 does not need to be conducted if reliable data are available causing developmental toxicity according to the criteria for classifying the test substance as R61. Therefore, a GLP-compliant prenatal developmental toxicity study with 2-MEA is not required. This is also in accordance with Regulation (EC) 1907/2006, which specifies that unnecessary tests should be avoided in terms of animal welfare reasons.
Reference not cited in the IUCLID:
CICAD (Concise Internation Chemical Assessment Document) 67, Selected Alkoxyethanols: 2 -Methoxyethanol, ISBN 92 4 153067 7, WHO, 2009
Justification for classification or non-classification
- Repr. Repr.1B – H360FD: “May damage fertility. May damage the unborn child”.
Harmonised classification:
2-methoxyethyl acrylate has been inserted into ATP15. The following classification applies:
Details:
Sexual function and fertility
In the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test performed in rat, the LOAEL for parental toxicity was 40 mg/kg bw/day based on histopathological changes in the testes and epididymis as well as atrophy, haemorrhage and apoptosis in the thymus. The LOAEL for reproductive effects was 40 mg/kg bw/day based on histopathological changes in the testis and epididymis and a dose-related increase in pre-coital time and reduced fertility at all dose levels.
Body weight and histopathological effects on reproductive organs were observed in the testis and epididymis from 40 mg/kg bw as follows:
- Body weight loss: At 250 mg/kg bw/day, most male animals and a few female animals showed a body weight loss, which slightly recovered during treatment at 150 mg/kg bw/day. Reduced body weight gains were also noted for males at 100 mg/kg bw/day.
The reduced body weight gains for females of the 100 mg/kg bw/day dose group during the first two weeks of post-coitum was considered a cause of their pregnancy status (i.e. implantation sites only instead of live foetuses) and not toxicologically relevant. However at 40 mg/kg bw/day, no indication of marked general toxicity has been observed.
- Mortality: At 250 mg/kg bw/day: 2 males died on day 2 (no cause of death could be determined), 1 male was killed on day 8 (showed ulcerative inflammation in the stomach with resultant peritonitis); at 100 mg/kg bw/day: 1 female killed in extremis on day 21 post-coitum.
No changes in body weights and mortality were observed at 40 mg/kg bw/day. Indeed, at this dose only changes in haematological parameters were observed in females (decreased MCV and MCH). The adversity of these findings is not clear as no change in haematocrit and haemoglobin was reported.
- Histopathology effects on reproductive organs: At 100 mg/kg bw/day: degeneration of seminiferous tubular epithelium, oedema, necrosis inflammation and enlarged ampholitic cells in testes, impairment of the spermatogenetic cycle in testis, sperm granuloma, degeneration, atrophy and inflammation in epididymis. At 40 mg/kg bw/day: necrosis, enlarged ampholitic cells, impairment of the spermatogenetic cycle in testis. There were no statistically significant changes in histopathological observations based on Fisher's Exact test at 5% or 1% level and on Steel’s test at 5%.
Fertility effects were observed at all dose levels including increase pre-coital time of females treated at 40 mg/kg bw/day and 250/150 mg/kg bw/day and dose-related decreased fertility index: at 100 mg/kg bw/day reduction of fertility index (90% calculated as no. animals with implants/no. of mating x 100) and reduction of number of corpora lutea and implantation sites. In addition, a dose related decrease was noted for number of corpora lutea (control group: 14.5; 11.3 and 9.9 at 40 and 100 mg/kg bw/day, respectively) and implantation sites (control group: 11.1; 9.8 and 7.8 at 40 and 100 mg/kg bw/day, respectively). Steel’s test significant at 1% level was observed only in “no. of corpora lutea” in the highest dose.
RAC concludes, that the available data on reproductive toxicity, dose-related fertility effects (increased precoital time, reduced fertility index, reduced number of corpora lutea and implantation sites) at all dose levels, histopathological changes in the testis and epididymis at all dose levels and statistically significant changes in reproduction organ weights of males, represent clear evidence of adverse effects on sexual function and fertility.
Fertility effects were not considered to be secondary non-specific consequences to the high parental toxicity observed at 100 and 250/150 mg/kg bw/day (body weight loss, mortality) since they were present at 40 mg/kg bw/day, where no indication of marked general toxicity was observed.
The effects on fertility were also supported by data from the primary metabolite 2-methyoxyethanol.
The available animal data support classification for reproductive toxicity category 1B H360F. There is no information that the effects may not be relevant to human and the quality of the study is good, therefore, RAC considers that a classification as Repr. 1B; H360F “May damage fertility” is warranted.
Developmental toxicity
In the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test performed in rat, dose-related decrease in live births and viability index was observed at all dose tested.
The observed maternal effects included a body weight loss at 250 mg/kg bw/day in a few female, which slightly recovered during treatment at 150 mg/kg bw/day; a body weight loss at the end of post-coitum for females treated with 100 mg/kg bw/day; the death of one female on day 21 post-coitum at 100 mg/kg bw/day. It is noticed that at 40 mg/kg bw/day, where no indication of marked general maternal toxicity was observed, a decrease in live births and in viability index were observed. At 100 and 150/250 mg/kg bw/day, where high maternal toxicity occurred, no dam had live pups on day 1. Since limited maternal toxicity was reported in the low dose group, the effects on development is not considered to be a secondary non-specific consequence of maternal toxicity.
In the opinion of RAC, due to marked developmental effects manifesting as dose-related decreases in live births and viability index at all doses, as observed in an OECD guideline-compliant developmental screening study, 2-methoxyethyl acrylate was considered to meet the criteria for classification as Repr. 1B; H360D “May damage the unborn child”. Since limited maternal toxicity was reported in the low dose group, the effects on development were not considered to be a secondary consequence of maternal toxicity.
The effects on development were also supported by data from the primary metabolite 2-methyoxyethanol.
There was no information that the effects could not be relevant for humans and therefore Repr. 2 was not considered appropriate.
Conclusion on fertility and development
The LOAEL for adverse effects on sexual function and fertility was 40 mg/kg bw/day based on:
- dose-related fertility effects (increase in precoital time, reduced fertility index, reduced number of corpora lutea and implantation sites) at all dose levels, but without clear dose-response relationships for all parameters - Steel’s test statistically significant at 1% level was observed only at the highest dose,
- histopathological changes in the testis and epididymis at all dose levels (not statistically significant based on Fisher's Exact test at 5% or 1% level and on Steel’s test at 5%) and
- statistically significant changes in reproductive organ weights of males (Dunnett’s test).
Fertility effects were not considered to be secondary non-specific consequences at the high parental toxicity observed at 100 and 250/150 mg/kg bw/day (body weight loss, mortality) since at 40 mg/kg bw/day where the effects were also observed, no indication of marked general toxicity has been observed.
The LOAEL for developmental toxicity was 40 mg/kg bw/day based on statistically significant (Fisher's Exact test significant at 1% level) decreased live litters and decrease viability index.
In summary, the decreased live litters and viability index observed in the developmental screening study were considered sufficient effects for classification as Repr. 1B; H360D “May damage the unborn child”, supported by evidence from the well documented reproductive toxicity data for the main metabolite, 2-methoxyethanol.In conclusion, based on marked fertility and developmental effects in animals, RAC is of the opinion that 2-methoxyethyl acrylate meets the criteria for classification as Repr. 1B; H360FD “May damage fertility. May damage the unborn child”.
Specific concentration limit
No specific concentration limit could be set for 2-methoxyethyl acrylate based on the limited data available from the screening study OECD TG 422 as no ED10 (effective dose with a 10% effect level above the background) could be determined in the available screening study (Guidance p. 3.7.2.5.1.).
Adverse effects on or via lactation
There is no information to propose a classification for effects on or via lactation. In the reproductive screening toxicity study, no pups were born at 100 and 250/150 mg/kg bw/day. Out of the nine litters at 40 mg/kg bw/day, only six had live pups. In addition, most of these pups did not survive the first days of lactation
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