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Description of key information

Repeated dose toxicity of HPMA has been investigated in an OECD 422 combined repeated dose and reproductive toxicity study. A chronic study does not have to be performed, because, like MMA, the substances are rapidly metabolised, and by analogy to MMA ultimately metabolised to carbon dioxide and water. Therefore, as demonstrated in the case of MMA in carcinogenicity studies of up to 2 years duration, there is no concern for lesions due to accumulative toxicity. MMA data with different species and different application routes are used by read-across. For PG, the glycolic metabolite of HPMA, long term feeding studies up to 2 years in rodent and non-rodent species are available. Renal effects were observed only in high dosages.

Read across evaluation according to ECHA’s ReadAcrossAssessment Framework (RAAF)

The metabolism from the HPMA to its primary metabolites is well understood. The same is true for the further metabolism pathways of MAA and the alcohol metabolite PG, respectively (see chapter 5.2, ATSDR 1997/ 2008/ 2010, NTP 2004a, 2004b). The endpoint specific “scientific assessment” of the read across is thus “acceptable with a high level of confidence”.

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP, japanese study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Early version of guidance for OECD 422 not containing functional observation battery test 
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: male 315 ~ 359 g; females 210 ~ 243 g,
- Fasting period before study: yes
- Housing: During the quarantine: suspended using a stainless steel cage type 1 with 5 per cage; Breeding: divided into separate rearing cages. Moved to a separate plastic cage, having had a natural birth .
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: five-day quarantine period and then set up a six-day acclimation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 deg C
- Humidity (%): 40-70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Concentration of samples was prepared by dissolving the required water for injection. The concentrations of preparation is protectedc from light at room temperature for 7 days to ensure that there were no stability issues. Preparation of 0.6% solution concentration is below the threshold level of determination, because we could not confirm the stability during the preparation for the 6% solution diluted with water for injection prepared in concentration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0.6, 0.8, 2.6 and 20%
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
Males :49 days
Females: from 14 days before mating to day 3 of lactation
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0 (vehicle), 30, 100, 300 and 1000 mg/kg/ day
Basis:
nominal in water
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 2-week preliminary study
- Rationale for animal assignment (if not random): random by weight
- Terminal kill: Males, day 50; Females, day 4 of lactation
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day
- Cage side observations: general condition

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 2 times per week
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:day after treatment
- Anaesthetic used for blood collection: Yes (identity) : sodium pentobarbital
- Animals fasted: No data
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day after treatment
- Animals fasted: No data
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: sexual cycle until confirmation; status of delivery

Sacrifice and pathology:
GROSS PATHOLOGY: Yes; Males: Thymus, liver, kidney, testis and epididymis weight was measured after removal, adrenal gland, brain, heart and spleen and 10% neutral buffered formalin solution (However, testicular and epididymal fluid Buan) was fixed; Females: counting the number of corpora lutea and the number of implantation scars. Liver, kidney, ovary and thymus weight was measured after removal, adrenal gland, brain, heart and spleen with a fixed 10% neutral buffered formalin solution.
HISTOPATHOLOGY: Yes; Control group and 1000 mg / kg group of heart, liver, spleen, thymus, kidney, testis, epididymis, ovary, adrenal and brain for the Preparation HE staining of tissue was examined histologically.
Statistics:
In either test, significant risk factors were less than 5%.
1) multiple comparison test
Weight (the parent animals, babies), food consumption, number of estrus, days mating, pregnancy [Day delivery (feeding 0) - date confirmed mating, the number of implantation scars, the number of birth control mobilize (number of babies stillborn baby + ), the number of newborn, number of children born dead, birth rate [(number of birth control mobilize / number of implantation scars) × 100], rate of production of child [(number of infant feeding 0 days / number of implantation scars) × 100], corpus number, implantation rates [(number of implantation scars / number of corpora lutea) × 100], fertility [(number of infant feeding 0 day / mobilize all of birth control) × 100], feeding baby number four day, feeding 4 day survival rate [(number of newborn feeding 4 days / 0 Number of infant feeding day) × 100], unusual occurrence rate [(number of children with abnormal/ number of newborns) × 100], sex ratio (male / female), organ weights ( including the relative weight), results of blood tests, blood biochemistry test results for the mean and standard deviation were calculated for each group. Significant difference test, Bartlett's test and the homoscedasticity of Law, Law-way layout analysis of variance if the variance 1) and, if significant Dunnett method 2) or Scheff Method 3) were using. However, if the variance was not approved, the analysis method using centrally located position (Kruskal-Wallis test of 4)) and a significant if you use the ranking method or Dunnett Scheff Method was used.
2) χ ^ 2 test
Copulation rate [(number of established animal mating / number of live animals) × 100], fertility [(number of female fertility / Establishment of animal mating) × 100], the birth rate [(number of female newborns / number of female fertility) × 100] is, χ ^ 2 using the test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
For the males, salivation, decrease in locomotor activity and ptosis were found in the 1000 mg/kg group, and 2 animals of the group died. Decrease in hematocrit, tendencies for decrease in RBC and hemoglobin, and increase in the relative liver weights were also found in the 1000 mg/kg group. 
For the females, salivation, decrease in locomotor activity and ptosis were found in the 1000 mg/kg group, and 1 animal died.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
clinical signs
haematology
mortality
organ weights and organ / body weight ratios
Critical effects observed:
not specified
Conclusions:
In an OECD 422 study in rats, the oral repeated dose NOAEL was determined to be 300 mg/kg based on body weights.
Executive summary:

According to the OECD test guidelines for combined repeated dose toxicity study with the reproductive/developmental toxicity screening test [OECD TG 422], SD (Crj: CD) rats (12 animals/dose/sex) were administrated with this substance by gavage at doses of 0 (vehicle; water for injection), 30, 100, 300, and 1,000 mg/kg bw/day (12 animal/dose/sex). Males were dosed for a total of 49 days beginning 14 days before mating, and females were dosed for total of 41 to 48 days starting from 14 days before mating to day 4 of lactation throughout the mating and pregnancy period.

At 1000 mg/kg bw/day for males, two of the 12 animals died. Salivation, decreases in locomotor activity, ptosis were observed. The decrease in hematocrit and the tendencies for decrease in erythrocyte (RBC) and hemoglobin were found in haematological examination. The statistically significant increase in the relative liver weights was observed in organ weight. At that for females, one of the 12 animals died. Salivation, decreases in locomotor activity, ptosis were observed. The other significant toxicological changes were not observed. 

At 0, 30, 100 and 300 mg/kg bw/day for both sexes, no animals died and no abnormal clinical changes were observed. Moreover, significant toxicological changes were not observed in body and organ weight, food consumption, haematological examination, blood chemical examination, necropsy, and histopathological examination. Therefore, no adverse effect was observed in both sexes. 

The NOAELs for the repeated dose toxicity are considered to be 300 mg/kg bw/day for both sexes.

Endpoint conclusion
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

Repeated dose toxicity: oral

As a part of the Japanese HPV program HPMA has been evaluated for repeat-dose toxicity in Sprague-Dawley rats in the OECD 422 combined repeat-dose developmental/ reproductive toxicity screening test. These studies were conducted by the Japan Ministry of Health and Welfare and were assigned a K-2, reliable with restriction reliability rating.

HPMA

 In the study for HPMA, male rats (12/group) were given daily gavage doses of 0 (vehicle), 30, 100, 300 or 1000 mg/kg for 50 days including pre-mating, mating and post-mating intervals. Females (12/group) were administered the same doses for two weeks prior to mating, during mating and gestation up until day 4 of lactation. (approximately 54 days depending upon time to conception). Animals were observed for clinical symptoms of intoxication daily and food consumption and body weight were monitored throughout the study. Blood samples were taken for hematological and clinical chemistry analysis at study termination. Thymus, liver, kidney, testes, epididymes, and ovaries were weighed. In addition to these tissues,adrenal gland, brain, heart and spleen were fixed in 10% neutral buffered formalin solution for subsequent staining and histopathological evaluation.

Two male and one female of 12 exposed animals died on study at the 1000 mg/kg dose level. Clinical symptoms of intoxication observed at 1000 mg/kg [the highest dose tested] included: salivation, decrease in locomotor activity and ptosis for both sexes. Liver weight was increased in males only at 1000 mg/kg/d, with minimal hepatocyte vacuolation as a histopathological correlate. Also in males, a decrease in RBCs was observed at 1000 mg/kg only. The NOAEL for these effects was considered to be 300 mg/kg for both males and females.

 

The short half-life of HPMA (as desribed in the metabolism chapter) implies that, under normal physiological conditions, the systemic exposure to HPMA as parent ester is irrelevant for the hazard assessment. This is especially true when the low level of electrophilic reactivity as potential mode of action of the parent esters is considered. The combination of the short half-life and the weak electrophilic reactivity of the parent esters means that toxicity profile upon repeated dosing is determined by the profiles of the primary metabolites (PG and MAA).

Primary Metabolites

MAA

Due to the corrosivity of MAA, a repeated dose study by the oral route has not been performed. In a guideline inhalation study, ten male and ten female Sprague Dawley rats per test group were whole body exposed to MAA vapour on 6 hours per working day for 90 days (65 exposures). The target concentrations were 20, 40, 100 and 350 ppm (corresponding to 72, 143, 358 and 1252 mg/m³). At the high dose of 350 ppm, equivalent to a body burden of 308 mg/kg/d in males and 377 mg/kg/d in females[1], the 90-day inhalation exposure of rats to MAA induced signs of general toxicity as indicated by decreased body weight, body weight gain, food consumption and transiently food efficiency in the high concentration male animals. At 350 ppm, the local irritating effect was marginal, indicated by the hypertrophy/hyperplasia of the respiratory epithelium in the nasal cavity of two female animals. Under the current test conditions, the no-observed adverse effect level (NOAEL) in this study is 100 ppm (equivalent to a body burden of 88 mg/kg/d in males and 96 mg/kg/d in females) for the male and female rats.

PG

There are several animal studies available with orally applied Propylene glycol (PG). From those, reliable chronic studies from the dog and the rat were selected for the IUCLID dataset of HPMA as representative long-term studies for a rodent and a non-rodent species (Weil et al. 1971; Gaunt et al. 1972). For completenss reasons, the dataset is amended with subacute studies from the specifically sensitive species cat. ATSDR (1997) summarised the findings of the overall dataset as follows: “Results from animal studies indicate that intermediate and chronic exposure to propylene glycol may lead to hemolysis of red blood cells. Increased numbers of Heinz bodies (sign of red blood cell degeneration) were observed in cats exposed orally to > 1,000 mg/kg of propylene glycol for 2, 5, and 17 weeks, respectively (..). Other studies indicate increased Heinz body formation and decreased RBC survival in kittens and adult cats ingesting > 1,000 mg/kg/day (…). These findings are further supported by results obtained in dogs after chronic oral exposure to 5,000 mg/kg/day (Weil et al. 1971). Red blood cell hemolysis was evidenced by decreased hemoglobin and hematocrit levels, and decreased total red blood cell counts. In rats, however, there were no changes in any of the hematological parameters after 2 years of chronic oral exposure to 2,500 mg/kg/day propylene glycol (Gaunt et al. 1972). These results indicate that there may be species differences with regard to the effect of propylene glycol on red blood cells.” Reason for the species specific effects may be the different clearance capacity of the glucuronic acid conjugate: “In most mammals, part of the absorbed propylene glycol is eliminated unchanged by the kidney, while another portion is excreted by the kidneys as a glucuronic acid conjugate. The amount of propylene glycol eliminated by the kidneys has been estimated for humans at 45%, for dogs at 55−88%, and for rabbits at 24−14.2%. Cats do not have the ability to produce the glucuronidated metabolite.” (NTP-CERHR 2004). As a consequence of the different metabolism, results from cat studies have to be considered more cautiously than from other animals. Moreover, it is unlikely to achieve toxic PG levels from HPMA exposure.

Repeated dose toxicity: inhalation

There are no relevant inhalation studies available on HPMA. A limited three-week inhalation study in rats at saturated VP (approx. 0.5 mg/L) showed no toxic effects.

See also discussion regarding “Route” below

Repeated dose toxicity: dermal

There are no relevant dermal studies available on HPMA.

Repeated dose toxicity: other routes

No relevant studies available.

Human information

There are no relevant data available.

Summary and discussion of repeated dose toxicity

Subacute (49 d) repeated dose studies are available for both, HEMA and HPMA. For the primary metabolites methacrylic acid/MMA, ethylene glycol and propylene glycol subchronic or chronic repeated dose data are available. For comparison reasons, results from rat studies are shown below.


 

Table11: Repeated dose toxicity - NOAEC/NOAEL summary

Route & effects type

HEMA

MW 130.14

HPMA

MW 144.17

MAA      MW 86.09

MMA

MW 100.12

EG

MW 62.07

PG

MW 76.09

Inhalation

Local effects

---

---

100 ppm

358 mg/m

25 ppm

208 mg/m³
*
   **

---

---

Inhalation

Systemic effects

---

---

100 ppm

358 mg/m

500 ppm

1040 mg/m³

---

---

 

 

 

 

 

 

 

Oral

Systemic effects

100 mg/kg/d

(gavage)

300 mg/kg/d

(gavage)

---

164 mg/kg/d

(drinking water)

600 mg/kg/d

(feeding)

≥2500 mg/kg/d

(feeding)

0.77 mmol/ kg/d

2.08 mmol/ kg/d

 

1.64 mmol/ kg/d

9.67 mmol/ kg/d

32.9 mmol/ kg/d

*

The departure point for the risk assessment is the SCOEL IOLV of 50 ppm, taking into account observations in humans

**

No time-dependency of the lesion was found in several studies between 6 hours and 2 years

 

HEMA and HPMA have been evaluated for repeat-dose toxicity by the oral route in the OECD 422 protocol. In this protocol, both males and females were given daily oral gavage doses of HEMA and HPMA up to the limit dose of 1000 mg/kg/day for approximately 50 consecutive days. Toxicity was achieved in these studies as demonstrated by reduced food consumption and body weight gain at 1000 mg/kg/d. In addition, for HEMA only (not found for HPMA), kidney toxicity particularly in male rats as indicated by elevated BUN levels in serum, elevated kidney weights and minimal histopathological changes were described in 1000 mg/kg group animals. For males dosed with HEMA, the NOAEL was 100 mg/kg/d while the NOAEL for females administered HEMA was 300 mg/kg/d. No target organ was identified for HPMA even at doses which depressed body weight gain and food consumption.

These results indicate a mild effect of HEMA on the kidney which is considered a target organ for HEMA at very high repeat oral doses that depressed body weight gain and food consumption. These mild changes in kidney after approximately 50 days exposure indicate a small likelihood that effects of greater severity at the same effect doses or effects at lower doses would be observed in 90 day studies. The male rat (and Sprague-Dawley rat in particular) is prone to the spontaneous development of kidney toxicity beginning as early as 90 days of age (Goldstein 1988; Masoro, 1989). Thus, the mild effects described upon administration of limit doses of HEMA to male Sprague-Dawley rats may represent the interaction of the material with initial stages of development of spontaneous kidney toxicity in these animals.

 

Primary metabolites

MAA/MMA

Further, MMA, a structural analogue to HEMA and HPMA, has been extensively evaluated in repeat-dose studies by both oral and inhalation routes as reviewed in the EU RAR (2002). Inhalation studies in rats and mice conducted by the US NTP and in addition an oral drinking water study in rats were of 2 years duration. In these studies observed toxicity was confined to decreases in food consumption and body weight gain.Furthermore, there appears to be no marked increase in sensitivity for this non-specific toxicity with time.No target organ, including the kidney, for MMA was identified in these extensive studies.

PG

PG demonstrates systemic effects with blood and kidney effects only being observed only well above 1000 mg/kg (ATSDR 1997/2008).

Route

As outlined earlier, the saturated vapour density is approx. 109 ppm (650 mg/m³) for HPMA equivalent to a maximum body burden of approx. 160 mg/kg/d for a standard inhalation study in rat. In practice, the saturated vapour density is a theoretical value that cannot be achieved in full because under the conditions of an inhalation study the equilibrium is not reached fast enough. In consequence, that means that a study could only be performed below the expected NOAEL. The existing inhalation screening study confirms that.

Discussion

As described previously the short half-life of the parent esters combined with their weak electrophilic reactivity mean that toxicity profile upon repeated dosing is determined by the primary metabolites (PG and MAA).

In the case of HPMA after repeated exposure demonstrates general signs of toxicity and body weight loss at high dose (Furuhashi et al. 1997). While for PG there are only slight systemic effects with blood and kidney effects only being observed well above 1000 mg/kg (ATSDR 1997/2008). The relevant NOAEL of HPMA for the derivation of the DNEL is 300 mg/kg bw/d.

The common primary metabolite MAA is metabolised via the TCA cycle predominantly being eliminated as CO2 and water. After repeated exposure (350 ppm/-477 mg/kg/d, 90 days) there are no apparent adverse systemic findings other than reduced food consumption and transiently food efficiency in the high concentration male animals resulting in decreased terminal body weight and body weight gain in these animals.

Hence, the toxicity profile and the underlying mode of action is consistent between HPMA and its two primary metabolites MAA and PG. The observed systemic toxicity profile for the parent ester predominantly reflects the toxicity of the primary metabolite MAA.

In contrast to HEMA, where there is a slight additional contribution to the overall toxicity from the more toxic glycol metabolite, this is not the case for HPMA, as propylene glycol is of very low systemic toxicity. In addition, HEMA has a lower NOAEL than HPMA and the primary metabolites of HPMA. Thus, data generated on HEMA can act as a conservative surrogate for HPMA.

As outlined earlier, the inalation route is not an appropriate route of exposure for the hydroxyalkyl methacrylates HEMA and HPMA, because it is very unlikely that toxicologically relevant concentrations could be reached using the inhalation route.

Taken together, results of repeat-dose studies in HPMA and results from studies on MMA as representative of the chemical class of short-chain alkyl esters of methacrylic acid and PG, the alcohol metabolite, indicate that studies of longer duration (90 days) are not critical for the assessment of HPMA, because the mode of action is known – non-specific systemic toxicity – and the metabolite data in longer term studies with MMA and MAA indicate that there is little or no progression of this effect after the duration of approx. 50d of the OECD 422 study. Therefore, further longer term studies may be waived.

Justification for classification or non-classification

HPMA is not classified for repeated dose toxicity. Clinical symptoms of intoxication observed at 1000 mg/kg [the highest dose tested].

Liver weight was increased in males only. Females exposed to 1000 mg/kg HPMA had an elevated incidence of malacia of the medulla oblongata, but this effect was not observed at lower doses and was not observed in males.

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