2-methoxyethyl acrylate

Endpoint summary

Description of key information

NOAEL (OECD 422, subacute, oral, rat): < 40 mg/kg bw/day

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

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

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 oral 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

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

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 highest dose level was lowered to 150 mg/kg bw/day for both sexes due to severe toxicity noted at 250 mg/kg bw/day.

Observations and examinations performed and frequency:

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment (males); due to effects on reproduction, the timing was different for females of the control and 40 mg/kg bw/day dose group (during lactation) compared to 100 and 250/150 mg/kg bw/day dose group (during post-coitum)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight for max. 20 h
- How many animals: 5 per sex and group
- Parameters checked: white blood cells (WBC), differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils); red blood cells; reticulocytes; red blood cell distribution width (RDW); haemoglobin; haematocrit; mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentration (MCHC); platelets; prothrombin time (PT); activated Partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment (males); due to effects on reproduction, the timing was different for females of the control and 40 mg/kg bw/day dose group (during lactation) compared to 100 and 250/150 mg/kg bw/day dose group (during post-coitum)
- Animals fasted: Yes, overnight for max. 20 h
- How many animals: 5 per sex and group
- Parameters checked: alanine aminotransferase (ALAT); aspartate aminotransferase (ASAT); alkaline phosphatase (ALP); total protein; albumin; total bilirubin; urea; creatinine; glucose; cholesterol; sodium; potassium; chloride; calcium; inorganic phosphate (Inorg. Phos.); bile acids

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males: during Week 4 of treatment; females: towards the end of the scheduled lactation period (all before blood sampling); due to the effects on reproduction, the timing was different for one animal of the 40 mg/kg bw/day group and all selected females of the 100 and 250/150 mg/kg bw/day group (during Days 23-26 post-coitum)
- Dose groups that were examined: all (5 animals per group)
- Battery of functions tested: grip strength / motor activity / other: hearing ability, pupillary reflex, static righting reflex

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 1 under “Any other information on materials and methods incl. tables”)
HISTOPATHOLOGY: Yes (Table 1 under “Any other information on materials and methods incl. tables”)

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.

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 “Details on results”

Mortality:

mortality observed, treatment-related

Description (incidence):

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 “Details on results”

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 (Week 1-2): reduced food consumption; 250/150 (Days 14-20) and 100 (Days 17-20) mg/kg bw/day: dose-related decrease in food consumption

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

250/150 mg/kg bw/day (m): decreased haemoglobin, MCH, MCHC and platelets; 250/150 and 100 mg/kg bw/day (f): decreased haemoglobin, MCV, MCH, MCHC and platelets; increased prothrombin time; 40 mg/kg bw/day (f): decreased MCV and MCH

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

250/150 and 100 mg/kg bw/day (m): decreased total protein and calcium; increased chloride; 250/150 mg/kg bw/day (f): decreased total protein

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

250/150 mg/kg bw/day (m): number of total movements was slightly decreased

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

100 and 250/150 mg/kg bw/day: dose-related reduction in absolute and/or relative weights of the thymus, testes, prostate, epididymides and seminal vesicles (m)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

250/150 and 100 mg/kg bw/day: reduced size of testes, epididymides and thymus; 250/150 mg/kg bw/day: irregular surfaces in fore-stomachs (non-glandular portion) and glandular stomach (m)

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 “Details on results”

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
-Unscheduled deaths: at 250 mg/kg bw/day, 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 WEIGHT GAIN: see Table 2 under “Any other information on results incl. tables”
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, no body weight gain of these animals during lactation could be determined.

FOOD CONSUMPTION
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.

HAEMATOLOGY
Several haematological parameters were affected by treatment with the test substance at all dose levels (see Table 3 under “Any other information on results incl. tables). Decreased levels of haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and platelets were noted for males treated at 250/150 mg/kg bw/day. For females, decreased levels of haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and platelets (not statistically significant), and increased prothrombin time were noted at 100 and 250/150 mg/kg bw/day. In addition, mean corpuscular volume and mean corpuscular haemoglobin were also decreased for females treated at 40 mg/kg bw/day. All other statistical significant changes were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution, and were attributed to the pregnancy/lactation status of the females or high control values.

CLINICAL CHEMISTRY
Clinical biochemistry parameters were affected by treatment with the test substance at 100 and 250/150 mg/kg bw/day (see Table 3 under “Any other information on results incl. tables). Decreased levels of total protein and calcium and increased levels of chloride were noted for males treated at 100 and 250/150 mg/kg. For females at 250/150 mg/kg bw/day, total protein concentration was also decreased. Individual animals of all groups showed very high bile acid values. In the absence of a dose response relationship, this was considered not toxicologically relevant. All other statistically significant changes were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution or were considered due to the pregnancy/lactation status of the females.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals tested. For motor activity, all groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period. The number of total movements was slightly decreased for the males treated at 250/150 mg/kg bw/day, however not statistically significant. Due to the test substance related effect on reproduction/development, data of motor activity for the females could not be evaluated correctly as measurements were performed with large litters for the control group, with a few pups/litter for the 40 mg/kg bw/day group, and without any pups for the 100 and 250/150 mg/kg bw/day group.

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. 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 and 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: NON-NEOPLASTIC
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 3 under “Any other information on results incl. tables”).
Abnormal findings in the stomach due to the corrosive properties of the test substance 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 3 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 3 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. Incidences of pathological findings in the spleen are summarised in Table 3 under “Any other information on results incl. tables”.
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 at 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.

Key result

Dose descriptor:

LOAEL

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

Effect level:

< 40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects were observed

Critical effects observed:

not specified

Table1. Body weight changes after treatment

Body weights	Test substance [mg/kg bw/day]
	0	40	100	250/150
	MALES
Pre-mating period
Day 8 (Week 2)	306 ± 16	298 ± 12	293 ± 13	274 ± 17**
Mating period
Day 1 (Week 1)	323 ± 19	312 ± 14	303 ± 14*	283 ± 13**
Day 8 (Week 2)	330 ± 24	321 ± 14	309 ± 16*	292 ± 16**
Day 15 (Week 3)	343 ± 27	333 ± 15	319 ± 16*	303 ± 17**
End of treatment	327 ± 26	317 ± 16	300 ± 17*	288 ± 15**
	FEMALES
Post-coitum
Day 11	237 ± 9	242 ± 14	226 ± 8	216 ± 6*
Day 14	246 ± 8	252 ± 15	230 ± 9**	222 ± 1*
Day 17	266 ± 10	265 ± 13	231 ± 10**	214 ± 4**
Day 20	297 ± 13	289 ± 13	224 ± 13**	205 ± 1**
End of treatment	216 ± 8	216 ± 19	201 ± 11	192 ± 7*

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 2. Results of clinical chemistry analysis and haematology

Parameters	Test substance [mg/kg bw/day]
	0	40	100	250/150
HGB [mmol/L]
males	9.8 ± 0.3	9.2 ± 0.6	9.1 ± 0.2	8.6 ± 0.6**
females	9.4 ± 0.2	9.3 ± 0.3	8.8 ± 0.5*	8.5 ± 0.3**
MCV [fL]
males	52.2 ± 0.9	50.1 ± 1.7	50.6 ± 1.4	50.7 ± 1.3
females	55.3 ± 3.0	51.3 ± 1.0*	51.8 ± 1.2*	52.6 ± 2.5
MCH [fmol]
males	1.13 ± 0.03	1.08 ± 0.05	1.08 ± 0.03	1.05 ± 0.03**
females	1.24 ± 0.04	1.13 ± 0.03**	1.09 ± 0.03**	1.11 ± 0.04**
MCHC [mmol/L]
males	21.69 ± 0.42	21.53 ± 0.70	21.35 ± 0.56	20.72 ± 0.62*
females	22.39 ± 0.75	22.07 ± 0.41	21.10 ± 0.42**	21.16 ± 0.36**
Platelets [10E+9/L]
males	771 ± 99	761 ± 79	672 ± 127	508 ± 52**
females	814 ± 108	800 ± 164	668 ± 105	623 ± 91
PT [s]
males	17.2 ± 1.0	18.4 ± 0.9	19.4 ± 1.0	19.6 ± 3.0
females	16.3 ± 0.8	17.3 ± 0.6	18.5 ± 0.4**	19.0 ± 1.0**
TP [g/L]
males	65.8 ± 2.3	66.2 ± 2.7	61.5 ± 2.5*	57.8 ± 2.4**
females	64.4 ± 3.6	62.5 ± 5.2	63.8 ± 3.9	57.7 ± 1.8*
Cl- [mmol/L]
males	102 ± 1	102 ± 1	105 ± 1**	104 ± 1
females	95 ± 2	99 ± 1**	103 ± 1**	104 ± 2**
Ca2+ [mmol/L]
males	2.66 ± 0.04	2.63 ± 0.03	2.55 ± 0.03**	2.58 ± 0.07*
females	2.79 ± 0.10	2.67 ± 0.14	2.73 ± 0.10	2.66 ± 0.08

HGB = haemoglobin; MCV = mean corpuscular volume; MCH = mean corpuscular haemoglobin; MCHC = mean corpuscular haemoglobin concentration; PT = prothrombin time; TP = total protein; CL- = chloride; Ca2+ = calcium; SD = standard deviation

*/** Dunnett−test based on pooled variance significant at 5% (*) or 1% (**) level

Table 3. Incidences of primary and secondary histopathological findings

Histopathological findings	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	-
STOMACH (males)	5	5	5	8
- Inflammation necrotising	-	-	-	1
- Adhesions	-	-	-	1
- Hyperplasia non-glandular	-	3	3	7
- Degeneration vacuol.	1	-	-	-
- Degeneration glandular epithelium	-	-	-	2
- Hyperkeratosis	-	4	5	7
- Chronic active inflammation	-	-	2	5
- Increased infiltrates	1	1	-	-
- Haemorrhage	-	-	-	2
- Edema	-	-	-	1
STOMACH (females)	5	5	6	5
- Hyperplasia glandular	-	1	-	-
- Hyperplasia non-glandular	-	2	6	5
- Hyperkeratosis	-	3	6	5
- Chronic active inflammation	-	1	4	5
LIVER (males)	5	5	5	8
- Congestion	-	-	1	-
- Necrosis hepatocellular	-	-	-	2
- Adhesions	-	-	-	1
- Infiltrate mononuclear	4	5	5	2
- hyperplasia Kupffer cells	-	-	-	1
LIVER (females)	5	5	6	5
- Necrosis hepatocellular	-	-	-	1
- Infiltrate mononuclear	2	3	4	1
- Haematopoiesis	2	1	1	2
SPLEEN (males)	5	5	5	8
- Hyperplasia lymphoid	-	-	1	-
- Haematopoiesis	5	5	-	2
- Increased pigment	-	-	-	2
- Inflammation subacute	-	-	-	1
- Inflammation mesentery	-	-	-	1
- Lymphoid atrophy	-	1	-	-
SPLEEN (females)	5	5	7	5
- Increased megakaryocytosis	-	-	1	-
- Fibrosis	-	-	-	1
- Haematopoiesis	5	3	3	3
- Increased pigment	-	-	2	4
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

Conclusions:

Based on the results of the study, the NOAEL for systemic effects was considered to be < 40 mg/kg bw/day due to overall effects, but, in particular, due to alterations in haematological parameters in females and histopathological findings in testes and epididymides. 

Executive summary:

A combined 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 test substance at dose levels of 40, 100 and 250/150 mg/kg bw/day in propylene glycol via oral gavage. Males were treated for a period of 31 -35 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 one male animal that was sacrificed 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 in males and females found dead at both dose levels included hunched posture, piloerection, and pale and lean appearance. In surviving animals, treatment-related clinical signs including hunched posture, piloerection, salivation and rales were also noted at 250 mg/kg bw/day, but disappeared during dosing at 150 mg/kg bw/day.

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. 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.

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 post-coitum for females treated with 100 mg/kg bw/day. No changes in body weights were observed at 40 mg/kg bw/day.

Reduced food consumption was noted for the first two weeks of treatment at 250 mg/kg bw/day for both sexes, which 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.

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.

At clinical chemistry, statistically significant changes in examined parameters were noted at 100 and 250/150 mg/kg bw/day. Decreased levels of total protein and calcium were noted for males treated at 100 and 250/150 mg/kg bw/day. For females at 250/150 mg/kg bw/day, total protein concentration was statistically significantly decreased. All other statistically significant changes in haematology or clinical chemistry were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution or were considered due to the pregnancy/lactation status of the females.

Neurobehavioural examination showed that hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals tested. For motor activity, all groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period. The number of total movements was slightly, but not statistically significantly decreased for males treated at 250/150 mg/kg bw/day.

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 gross pathology, testes and epididymides of reduced size (with or without flaccidity) were present in males at 100 and 250/150 mg/kg bw/day. Due to corrosive properties of the test substance, fore-stomachs (non-glandular portion) had irregular surfaces in males and females at 250/150 mg/kg bw/day. This observation was also noted in the glandular stomach of some males treated with 250/150 mg/kg bw/day. Individual animals (males and females) at 100 mg/kg bw/day and some males at 250/150 mg/kg bw/day also had reduced thymus size, however corresponded to atrophy of cortical lymphocytes. 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 sacrificed in extremis revealed findings in the uterus, cervix, spleen, mandibular lymph nodes, upper jaw, and abdominal cavity. 

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. Testes observations included degeneration of seminiferous tubular epithelium, increased severity of edema, chronic active inflammation, and enlarged amphophilic cells. PAS stained testes observations included enlarged spermatagonia, asynchronous tubules, individual cell necrosis, spermatidic giant cells, and reduced layers of spermatagonia. At the highest dose level there were no recognizable stages. Epididymal observations included degenerated sperm, hypospermia, atrophy and inflammation. 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 addition, one female treated at 100 mg/kg, which was sacrificed in extremis showed suppurative inflammation in the uterus and abscessing inflammation in the jaw area. Based on the results of the study, the NOAEL for systemic effects was considered to be < 40 mg/kg bw/day due to overall effects, but, in particular, due to alterations in haematological parameters in females and histopathological findings in testes and epididymides. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

40 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

male reproductive system

Organ:

cauda epididymis

testes

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A combined 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) and in compliance with GLP.

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 one male animal that was sacrificed 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 in males and females found dead at both dose levels included hunched posture, piloerection, and pale and lean appearance. In surviving animals, treatment-related clinical signs including hunched posture, piloerection, salivation and rales were also noted at 250 mg/kg bw/day, but disappeared during dosing at 150 mg/kg bw/day.

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. 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.

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 post-coitum for females treated with 100 mg/kg bw/day. No changes in body weights were observed at 40 mg/kg bw/day.

Reduced food consumption was noted for the first two weeks of treatment at 250 mg/kg bw/day for both sexes, which 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.

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.

At clinical chemistry, statistically significant changes in examined parameters were noted at 100 and 250/150 mg/kg bw/day. Decreased levels of total protein and calcium were noted for males treated at 100 and 250/150 mg/kg bw/day. For females at 250/150 mg/kg bw/day, total protein concentration was statistically significantly decreased. All other statistically significant changes in haematology or clinical chemistry were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution or were considered due to the pregnancy/lactation status of the females.

Neurobehavioural examination showed that hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals tested. For motor activity, all groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period. The number of total movements was slightly, but not statistically significantly decreased for males treated at 250/150 mg/kg bw/day.

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 gross pathology, testes and epididymides of reduced size (with or without flaccidity) were present in males at 100 and 250/150 mg/kg bw/day. Due to corrosive properties of the test substance, fore-stomachs (non-glandular portion) had irregular surfaces in males and females at 250/150 mg/kg bw/day. This observation was also noted in the glandular stomach of some males treated with 250/150 mg/kg bw/day. Individual animals (males and females) at 100 mg/kg bw/day and some males at 250/150 mg/kg bw/day also had reduced thymus size, however corresponded to atrophy of cortical lymphocytes. 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 sacrificed in extremis revealed findings in the uterus, cervix, spleen, mandibular lymph nodes, upper jaw, and abdominal cavity. 

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. Testes observations included degeneration of seminiferous tubular epithelium, increased severity of edema, chronic active inflammation, and enlarged amphophilic cells. PAS stained testes observations included enlarged spermatagonia, asynchronous tubules, individual cell necrosis, spermatidic giant cells, and reduced layers of spermatagonia. At the highest dose level there were no recognizable stages. Epididymal observations included degenerated sperm, hypospermia, atrophy and inflammation. 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 addition, one female treated at 100 mg/kg, which was sacrificed in extremis showed suppurative inflammation in the uterus and abscessing inflammation in the jaw area. Based on the results of the study, the NOAEL for systemic effects was considered to be < 40 mg/kg bw/day due to overall effects, but, in particular, due to alterations in haematological parameters in females and histopathological findings in testes and epididymides. 

Justification for classification or non-classification

Harmonised classification :

In the opinion proposing harmonised classification and labelling at EU level of 2-methoxyacrylate (CLH-O-0000001412-86-202/F), RAC concluded that some level of toxicity after repeated dose exposure was observed, but not sufficient to fulfil the criteria for classification for repeated dose toxicity and therefore no classification is warranted for STOT RE.

 

There is quite limited information available on effects after repeated exposure to 2-methoxyethyl acrylate and no 90-day repeated-dose toxicity studies are available in the CLH report.

In the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422, GLP 1 - reliable without restriction) Wistar rats (10/sex/group) were exposed 7 days/week by gavage, 2 weeks prior to mating, during mating, and, for males, up to termination, for females, also during post-coitum at least 4 days of lactation. Dose levels were 0, 40, 100, 250/150mg/kg bw (250 mg from day 1 to 11; 150 mg from day 12 to study termination; dose reduced due to severe toxicity).

 

 

Sacrifice of all surviving males (after completion of the mating period) and females which delivered, on lactation days 5-7, and females which did not deliver 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).

Pronounced lethality of parental animals, about 30%, was observed at the beginning of exposure at 250 mg/kg bw/day (very close to acute oral LD50 of 404 mg/kg bw): two males died on day 2 and 1 male was killed on day 8 because of peritonitis. At 100 mg/kg bw/day: 1 female 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. Hepatocellular necrosis was observed, but only at the high dose level (250/150 mg/kg bw/day), i.e. very close to median lethal dose.

In addition, there were the following minimal or slight histopathological changes in the thymus in parental animals at all dose levels. In males, lymphoid cortical atrophy was present in 1/4 at 100 mg/kg bw/day and 4/10 at 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. These changes were not statistically significant (Fisher’s Exact test and the Steel’s test was applied to frequency data).

Furthermore, corrosion in the forestomach was described in the Comet assay performed with 2-methoxyethyl acrylate on male rats after two days oral exposure to 240 mg/kg/day (see mutagenicity section below), but histological examination of the liver was not provided.

Taking to account all above available evidence, RAC considers that some level of toxicity after repeated dose exposure was observed, but not sufficient to fulfil the criteria for classification for repeated dose toxicity and therefore no classification is warranted for STOT RE.

 

Self-classification :

Based on the above assessment, no additional self-classification is proposed.